Novel Structurally Designed shRNAs

ABSTRACT

Provided is an improved design of shRNA based on structural mimics of miR-451 precursors. These miR-451 shRNA mimics are channeled through a novel small RNA biogenesis pathway, require AGO2 catalysis and are processed by Drosha but are independent of DICER processing. This miRNA pathway feeds active elements only into Agog because of its unique catalytic activity. These data demonstrate that this newly identified small RNA biogenesis pathway can be exploited in vivo to produce active molecules.

This application is a continuation of U.S. Ser. No. 14/630,419, filedFeb. 24, 2015, now allowed, which is a continuation of U.S. Ser. No.13/642,802, filed Mar. 7, 2013, now U.S. Pat. No. 8,993,532, issued Mar.31, 2015, which is a §371 national stage of PCT InternationalApplication No. PCT/US2011/033615, filed Apr. 22, 2011, claiming thebenefit of U.S. Provisional Application No. 61/327,510, filed Apr. 23,2010, the contents of each of which are hereby incorporated by referencein their entirety.

This patent disclosure contains material which is subject to copyrightprotection, the copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

Throughout this application, patent applications, published patentapplications, issued and granted patents, texts, and literaturereferences are cited. For the purposes of the United States and otherjurisdictions that allow incorporation by reference, the disclosures ofthese publications are incorporated by reference into this application.

This invention was made with government support under GM062534 andCA013106 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“170306_82764-AAA-PCT-US_Substitute_Sequence Listing_AC.txt,” which is8.03 kilobytes in size, and which was created Mar. 6, 2017 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Mar. 6, 2017 aspart of this application.

1. BACKGROUND OF THE INVENTION

This invention relates in part to improvements directed to use of RNAinterference (RNAi) technology that exploits a newly identified smallRNA biogeneisis pathway.

Traditional RNAi technology in mammals takes advantage of the canonicalmicroRNA (miRNA) pathway. Starting with PolII transcribed precursorRNAs, the biogenesis pathway involves two steps: DROSHA/DGCR8 cleavesthe precursor transcript into a short hairpin RNA that is exported intothe cytoplasm and then processed by DICER RNAseIII enzyme to yield amature small (21-22 nt) RNA duplex, that is then loaded into one of fourArgonaute proteins (AGO1 through AGO4) to form an active RNA InducedSilencing Complex (RISC). The conventional endogenous RNAi pathwaytherefore comprises three RNA intermediates: a long, largelysingle-stranded primary miRNA transcript (pri-mRNA); a precursor miRNAtranscript having a stem-and-loop structure and derived from thepri-mRNA (pre-miRNA); and a mature miRNA.

Argonaute proteins are the key effectors of small RNA-mediatedregulatory pathways that modulate gene expression, regulate chromosomestructure and function, and provide an innate immune defense againstviruses and transposons (Hutvagner, G. & Simard, M. J. Nat Rev Mol CellBiol 9, 22-32 (2008)). The structure of Ago proteins is well conserved,consisting of an amino-terminal domain, the mid domain, and theirsignature PAZ and Piwi domains. Structure-function relationships in thisfamily are becoming increasingly well understood (Joshua-Tor, L. ColdSpring Harb Symp Quant Biol 71, 67-72 (2006)). The PAZ and Mid domainshelp to anchor the small RNA guide, with PAZ binding the 3′ end using aseries of conserved aromatic residues and the Mid domain providing abinding pocket for the 5′ end. The Piwi domain contains an RNAse H motifthat was cryptic in the primary sequence but easily recognizable in thetertiary structure. Loading of a highly complementary target into an Agobrings the scissile phosphate, opposite nucleotides 10 and 11 of thesmall RNA guide, into the enzyme active site, allowing cleavage of theRNA to leave 5′ P and 3′ OH termini (Elbashir, S. et al. Genes Dev 15,188-200 (2001), Elbashir, S. M., et al. EMBO J 20, 6877-88 (2001), Yuan,Y. R. et al. Mol Cell 19, 405-19 (2005), Martinez, J. & Tuschl, T. GenesDev 18, 975-80 (2004), Schwarz, D. S., et al. Curr Biol 14, 787-91(2004)).

Ago proteins can be divided into three clades. The Piwi clade is animalspecific, and forms part of an elegant innate immune system thatcontrols the activity of mobile genetic elements (Malone, C. D. &Hannon, G. J. Cell 136, 656-68 (2009)). The Wago clade is specific toworms and acts in a variety of different biological processes (Yigit, E.et al. Cell 127, 747-57 (2006)). The Ago clade is defined by similarityto Arabidopsis Ago1 (Bohmert, K. et al. EMBO J 17, 170-80 (1998)).Ago-clade proteins are found in both plants and animals where oneunifying thread is their role in gene regulation. In plants, some Agofamily members bind to microRNAs and are directed thereby to recognizeand cleave complementary target mRNAs (Baumberger, N. & Baulcombe, D. C.Proc Natl Acad Sci USA 102, 11928-33 (2005), Qi, Y., Denli, A. M. &Hannon, G. J. Mol Cell 19, 421-8 (2005)).

Animal microRNAs function differently from their plant counterparts,with nearly all microRNA-target interactions providing insufficientcomplementarity to properly orient the scissile phosphate for cleavage.Here, target recognition relies mainly on a “seed” sequencecorresponding to miRNA nucleotides (Joshua-Tor, L. Cold Spring Harb SympQuant Biol 71, 67-72 (2006), Malone, C. D. & Hannon, G. J. Cell 136,656-68 (2009)). While pairing of the target to other parts of the miRNAcan contribute to recognition, seed pairing appears to be the dominantfactor in determining regulation (Yekta, S. et al. Science 304, 594-6(2004)). A very few extensive microRNA-target interactions can lead totarget cleavage in mammals (Davis, E. et al. Curr Biol 15, 743-9 (2005),Harfe, B. D. et al., Proc Natl Acad Sci USA 102, 10898-903 (2005)).However, none of these has yet been shown to be critical for targetregulation (Sekita, Y. et al. Nat Genet 40, 243-8 (2008), Hornstein, E.et al. Nature 438, 671-4 (2005), Tolia, N. H. & Joshua-Tor, L. Nat ChemBiol 3, 36-43 (2007)).

Despite the fact that animal microRNAs regulate targets withoutAgo-mediated cleavage, the Argonaute catalytic center is deeplyconserved. This consists of a catalytic DDH triad that serves as a metalcoordinating site (Liu, J. et al. Science 305, 1437-41 (2004)). Of thefour Ago-clade proteins in mammals, only Ago2 has retained both the DDHmotif and demonstrable endonuclease activity (Rivas, F. V. et al. NatStruct Mol Biol 12, 340-9 (2005), Song, J. et al. Science 305, 1434-7(2004), Azuma-Mukai, A. et al. Proc Natl Acad Sci USA 105, 7964-9(2008)). Ago1, Ago3, and Ago4 are linked within a single ˜190 kb locusand have lost catalytic competence. An analysis of Ago2 mutant cells hasindicated that proteins encoded by the Ago 1/3/4 locus can supportmiRNA-mediated silencing (Rivas, F. V. et al. Nat Struct Mol Biol 12,340-9 (2005)). This leaves us without a clear explanation for themaintenance of a catalytically competent Ago family member, since miRNAsare the exclusive partners of these proteins in almost all cell types(Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P. & Blelloch, R.,Genes Dev 22, 2773-85 (2008); Ender, C. et al. Mol Cell 32, 519-28(2008) Tam, O. C. et al. Nature, 453:534-538(2008); Kaneda, M. et al.,Epigenetics Chromatin, 2:9 (2009)).

2. SUMMARY OF THE INVENTION

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

Provided is an improved design of shRNA based on structural mimics ofmiR-451 precursors. These miR-451 shRNA mimics are channeled through anovel small RNA biogenesis pathway, require AGO2 catalysis and areprocessed by Drosha but are independent of DICER processing. This miRNApathway feeds active elements only into Ago2 because of its uniquecatalytic activity. These data demonstrate that this newly identifiedsmall RNA biogenesis pathway can be exploited in vivo to produce activemolecules.

Use of miR-451 shRNA mimics provides a distinct advantage overconventional shRNAs in that only one active strand is generated, therebyeliminating off-target effects that could result from incorporation ofthe sense strand of the duplex into an active RISC. The design of amiR-451 shRNA mimic is very simple and does not require use of sequencesfrom the miR-451 precursor molecule as it is only a structural mimic.However, parts of the primary miRNA-451 sequence, or of the primarysequence of another miRNA may be used in some embodiments, e.g., inaspects of the invention relating to primary shRNA mimics. In someaspects of the invention, primary miR-451 mimics are processed by thedrosha step of miR-451 processing while bypassing the canonical pathway,e.g. in certain shRNAs of the invention that are loaded into Ago2directly.

In one aspect, the invention provides for design and use of miR-451shRNA mimics based on existing siRNA molecules. In another aspect, theinvention provides for design and use of miR-451 shRNA mimics based onany 21-23 nt sequence in the coding region of a target gene. In anotheraspect, the invention provides for design and use of miR-451 shRNAmimics based on any 21-23 nt sequence in the non-coding region of atarget gene. In particular, the miR-451 shRNA mimic comprises a sequencethat is fully complementary to a 21 to 23 nucleotide long sequence inthe target gene, or to the 21 to 23 nucleotide target sequence of thesiRNA. In another aspect of the invention, the miR-451 shRNA mimiccomprises a sequence that is fully complementary to a 15 nucleotide longsequence in the target gene, or to a 15 nucleotide target sequence ofthe siRNA, wherein at least three nucleotides of the guide strand of themiR-451 mimic are in the loop of the shRNA hairpin. In another aspect ofthe invention, the miR-451 shRNA mimic comprises a sequence that isfully complementary to a 16 nucleotide long sequence in the target gene,or to a 16 nucleotide target sequence of the siRNA, wherein at leastthree nucleotides of the guide strand of the miR-451 mimic are in theloop of the shRNA hairpin. In designing the miR-451 shRNA mimic, thisfully complementary sequence is positioned within the shRNA, such thatAgo2 processing of the shRNA and further trimming within the RISCcomplex generates an active silencing molecule comprising said fullycomplementary sequence.

In some embodiments, the shRNA of the invention is a synthetic shRNA.

In a non-limiting example, design of a miR-451 mimic shRNA targeting p53is depicted in (FIG. 3). The resulting ˜40 nt shRNA has a short stem anda tight loop and cannot be processed by DICER. Instead, it is cleaved byAGO2 and then further trimmed to generate the active strands targetingp53 mRNA. In another non-limiting example described herein below inExample 6, p53 may be knocked down using the primary sequence backboneof miR-451 by grafting a p53-targeting shRNA sequence into the primarysequence of miR-451.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 19, 20 or 21 nucleotides fully complementary to a sequencein a target gene, having a sequence other than the mature sequence ofmiR-451, and a second sequence directly following the first sequence,wherein the second sequence is fully complementary to the sequence ofthe first 15 or 16 nucleotides counted from the 5′ end of the firstsequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides complementary to a sequence in atarget gene, and a second sequence directly following the firstsequence, wherein the second sequence is fully complementary to thesequence of the first 17 nucleotides counted from the 5′ end of thefirst sequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence iscomplementary to the sequence of the first 17 nucleotides counted fromthe 5′ end of the first sequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides complementary to a sequence in atarget gene, having a sequence other than the mature sequence ofmiR-451, and a second sequence directly following the first sequence,wherein the second sequence is complementary to the sequence of thefirst 17 nucleotides counted from the 5′ end of the first sequence.

In one aspect of the invention, an shRNA is provided having thestructure

wherein X₂ to X₂₂ are nucleotides complementary to a sequence in atarget gene, and are in a sequence other than the mature sequence ofmiR451; Y₄ to Y₂₀ are nucleotides complementary to X₂ to X₁₈; and X₁,Y₁, Y₂, and Y₃, are nucleotides that may be present or absent, wherein,X₁ and Y₃, when present, may be complementary or not complementary.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence is fullycomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence.

In various embodiments of the instant shRNA, the last 3 nucleotides, oralternatively the last 4 nucleotides, of the first sequence form a loopregion in the short hairpin molecule.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 3′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 3′ end.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 5′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 5′ end.

In an embodiment of the instant shRNA, the shRNA has no 3′ or 5′overhang.

In an embodiment of the instant shRNA, the shRNA consists of a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence is fullycomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein an intron or other non-coding region of a target gene, and a secondsequence directly following the first sequence, wherein the secondsequence is fully complementary to the sequence of the first 17 or 18nucleotides counted from the 5′ end of the first sequence.

In some embodiments of the invention, an shRNA comprises a firstsequence of 21, 22, or 23 nucleotides fully complementary to a sequencein a non-coding target gene.

In some embodiments of the invention, an shRNA comprises a firstsequence of 21, 22, or 23 nucleotides fully complementary to a sequencein a target long non-coding RNA.

In various embodiments of the instant shRNA, the last 3 nucleotides, oralternatively the last 4 nucleotides, of the first sequence form a loopregion in the short hairpin molecule.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 3′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 3′ end.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 5′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 5′ end.

In an embodiment of the instant shRNA, the shRNA has no 3′ or 5′overhang.

In an embodiment of the instant shRNA, the shRNA consists of a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence is fullycomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence.

Other aspects of the invention include an expression vector comprising asequence encoding an shRNA as described herein, operably linked to anRNA polymerase promoter, and a library of such expression vectors. Theexpression vector or library of expression vectors can be introducedinto a mammalian cell in vitro or in vivo in a method of attenuatingtarget gene expression. The shRNA is expressed in an amount sufficientto attenuate target gene expression in a sequence specific manner. In apreferred embodiment, the shRNA is stably expressed in the mammaliancell.

In one aspect of the invention a method is provided for attenuatingexpression of a target gene in a mammalian cell, the method comprisingintroducing into the mammalian cell an expression vector comprising asequence encoding a short hairpin RNA molecule (shRNA) operably linkedto an RNA polymerase promoter, the shRNA comprising:

(i) a first sequence of 21, 22 or 23 nucleotides fully complementary toa sequence in the coding region of the target gene,

(ii) a second sequence directly following the first sequence, whereinthe second sequence is fully complementary to the sequence of the first17 or 18 nucleotides counted from the 5′ end of the first sequence,

wherein the shRNA molecule is expressed in the mammalian cell in anamount sufficient to attenuate expression of the target gene in asequence specific manner, whereby expression of the target gene isinhibited.

In certain embodiments, the instant expression vector comprises asequence encoding the shRNA according operably linked to an RNApolymerase promoter. In certain embodiments, the invention provides foruse of a library of expression vectors, wherein each expression vectorcomprises a sequence encoding the shRNA operably linked to an RNApolymerase promoter.

In another method of attenuating target gene expression, the shRNA ofthe invention is introduced into a mammalian cell in an amountsufficient to attenuate target gene expression in a sequence specificmanner. The shRNA of the invention can be introduced into the celldirectly, or can be complexed with cationic lipids, packaged withinliposomes, or otherwise delivered to the cell. In certain embodimentsthe shRNA can be a synthetic shRNA, including shRNAs incorporatingmodified nucleotides, such as those with chemical modifications to the2′—OH group in the ribose sugar backbone, such as 2′-O-methyl (2′OMe),2′-fluoro (2′F) substitutions, and those containing 2′OMe, or 2′F, or2′-deoxy, or “locked nucleic acid” (LNA) modifications. In someembodiments, an shRNA of the invention contains modified nucleotidesthat increase the stability or half-life of the shRNA molecule in vivoand/or in vitro. Alternatively, the shRNA can comprise one or moreaptamers, which interact(s) with a target of interest to form anaptamer:target complex. The aptamer can be at the 5′ or the 3′ end ofthe shRNA. Aptamers can be developed through the SELEX screening processand chemically synthesized. An aptamer is generally chosen topreferentially bind to a target. Suitable targets include small organicmolecules, polynucleotides, polypeptides, and proteins. Proteins can becell surface proteins, extracellular proteins, membrane proteins, orserum proteins, such as albumin. Such target molecules may beinternalized by a cell, thus effecting cellular uptake of the shRNA.Other potential targets include organelles, viruses, and cells.

Also included in the invention is an isolated mammalian cell comprisingthe shRNAs described herein. In a preferred embodiment, the mammaliancell is a human cell. Another aspect of the invention provides anon-human mammal comprising the cell described above. In certainembodiments, the non-human mammal may be a chimeric mammal, some ofwhose somatic or germ cells comprising the shRNAs described herein.Alternatively, the non-human mammal may be a transgenic mammal, all ofwhose somatic or germ cells comprise the shRNAs described herein. Thus,transgenic mammals whose genomes comprise a sequence encoding the shRNAsof the invention are also provided. In one embodiment, the transgenicmammal is a mouse.

Also included in the invention is an isolated non-mammalian cellcomprising the shRNAs described herein. The cells may be those ofvertebrate organisms, or non-vertebrate organisms such as insects. Thecells may be those of fish (e.g. those of the Fugu genus, or the Daniogenus), frogs (e.g. those of the Xenopus genus), round worms (e.g. thoseof the Caenorhabdis genus), flies (such as the Drosophila genus), orothers. Another aspect of the invention provides a non-human animalcomprising the cell described above. In certain embodiments, thenon-human animal may be a chimeric animal, some of whose somatic or germcells comprising the shRNAs described herein. Alternatively, thenon-human animal may be a transgenic animal, all of whose somatic orgerm cells comprise the shRNAs described herein. Thus, transgenicanimals whose genomes comprise a sequence encoding the shRNAs of theinvention are also provided.

Another aspect of the invention provides for design of miRNAs based onstructural mimics of miR-451 precursors. In certain embodiments, suchstructural miRNA mimics of miR-451, or an expression vector or libraryof expression vectors encoding such structural mimics can be introducedinto different genetic backgrounds of mammalian cells, in particular incells deficient of the canonical pathway enzymes, such as dicer, toscreen for and identify such miRNAs capable of rescuing the nullphenotype and the functional roles of such miRNAs in contributing to thephenotype.

In one aspect of the invention, a non-naturally occurring miRNA isprovided comprising a first sequence of 21, 22 or 23 nucleotidescorresponding to the entire mature sequence, or a portion of thatsequence, of a mammalian miRNA other than miR-451, and a second sequencedirectly following the first sequence, wherein the second sequence isfully complementary to the sequence of the first 17 or 18 nucleotidescounted from the 5′ end of the first sequence. The skilled practitionerwill appreciate that the mature sequence of a mammalian miRNAalternatively may be identified as the sequence of the guide strand, orguide sequence for that miRNA.

In various embodiments of the instant non-naturally occurring miRNA, thelast 3 nucleotides, or alternatively the last 4 nucleotides, of thefirst sequence form a loop region in the short hairpin molecule.

In various embodiments of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has a 1 nucleotide overhang at its 3′ end,or alternatively a 2, 3 or more than 3 nucleotide overhang at its 3′end.

In various embodiments of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has a 1 nucleotide overhang at its 5′ end,or alternatively a 2, 3 or more than 3 nucleotide overhang at its 5′end.

In an embodiment of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has no 3′ or 5′ overhang.

An aspect of the invention provides a composition comprising an shRNAcomprising a first sequence of 21, 22 or 23 nucleotides fullycomplementary to a sequence in the coding region of a target gene, and asecond sequence directly following the first sequence, wherein thesecond sequence is fully complementary to the sequence of the first 17or 18 nucleotides counted from the 5′ end of the first sequence.

An aspect of the invention provides a pharmaceutical compositioncomprising an shRNA comprising a first sequence of 21, 22 or 23nucleotides fully complementary to a sequence in the coding region of atarget gene, and a second sequence directly following the firstsequence, wherein the second sequence is fully complementary to thesequence of the first 17 or 18 nucleotides counted from the 5′ end ofthe first sequence.

An aspect of the invention provides an shRNA comprising a first sequenceof 21, 22 or 23 nucleotides fully complementary to a sequence in thecoding region of a target gene, and a second sequence directly followingthe first sequence, wherein the second sequence is fully complementaryto the sequence of the first 17 or 18 nucleotides counted from the 5′end of the first sequence for the manufacture of a medicament.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1D show use of miR-451 shRNA mimics for RNAi in mammaliancells. Schematic depictions of the pre-let-7c-miR-451 mimic hairpin (SEQID NO: 1) compared to the native pre-let-7c (SEQ ID NO: 2). Guide strandin red and passenger strand in blue. (FIG. 1A) Overlapping GFPhistograms reporting the activity of let-7c molecules using GFP let-7csensor ES cells. Cells were co-transfected with PE-siRNA control. 105 PEpositive cells were gated and analyzed for GFP expression. (FIG. 1B)Dual luciferase assay reporting mature let-7 activity. Top: Schematic oflet-7 MSCV-luciferase reporter construct containing two perfectlymatching let-7c sites at the 3′UTR. Bottom: Histogram showingluminescence values of luciferase/renilla ratios. Data are the mean ofthree technical replicates ±SD. (FIG. 1C) Top panel: schematic of thep53 hairpin design in the mir-30 backbone (SEQ ID NO: 3) or mimickingthe miR-451 fold (SEQ ID NO: 4). Bottom panel: Western blot analysisshowing p53 knockdown in ES cells upon transfection of p53 hairpins andinduction of p53 with adriamycin. (FIG. 1D)

FIG. 2A-FIG. 1B show steps in the biogenesis of miR-451shRNA. Model ofmiR-451 biogenesis using an artificial MSCV expression plasmid. Theprimary transcript, driven by the LTR promoter, is processed by droshato release the 40 nt pre-miR-451 (SEQ ID NO: 5) that is processed byAgo2 to generate the active RISC complex. (FIG. 2A) IP-northerns showingprocessing of the mature miR-451 only in the Ago2 immunoprecipitates.Ago1 complexes could load pre-miR-451 but were unable to process it toits mature form. (FIG. 2B)

FIG. 3A-FIG. 3B show a schematic example for generating a miR-451 mimicmolecule. Here p53-shRNA-1224 is shown as an example targeting thefollowing site in the p53 message:UCCACUACAAGUACAUGUGUAA (SEQ ID NO: 6).FIG. 3A depicts the canonical miRNA processing pathway using the mir-30backbone of the p53 hairpin (SEQ ID NO: 7). The mir-30 loop sequence ingreen. The strands are color coded: antisense strand in red and sensestrand corresponding to the target site in blue. DICER RNAseIII cutsites are depicted using arrows. FIG. 3B shows the generation of thep53-1224 shRNA mimicking miR-451 structure that can be channeled throughthe Ago2 mediated miRNA biogenesis pathway. The antisense strand (red)spanning the stem is designed to extend into the loop. The passenger armis highlighted in blue. Ago2 catalysis of the predicted phosphodiesterbond is shown using scissors. This pathway generates only Ago2 activeRISC.

FIG. 4A-FIG. 4C show mature miR-451 expression depends on Ago2catalysis. Scatter plot of miRNA reads in wild-type versus mutant fetalliver. (FIG. 4A). Quantitative RT-PCR of primary transcript levels ofmir-144 (SEQ ID NO: 8) and miR-451 (SEQ ID NO: 9) in wt and mutant liversamples. Data are the mean of three biological replicates+/−SD*t-testwith equal variance p>0.05. (FIG. 4B). The unique structure of themiR-451 hairpin compared to mir-144 with the mirbase annotation ofmature miR-451 and mir-144 mapped to the predicted secondary hairpinstructure shown. Guide strand in red and passenger strand in blue. (FIG.4C).

FIG. 5A-FIG. 5F show the non-canonical biogenesis of miR-451. Effect onmature miRNA levels of Drosha conditional ablation in Drosha flox/floxCre-ER MEFs. (FIG. 5A). In-vitro processing of miR-451 and mir-144primary transcripts by Drosha immunoprecipitates. pre-miR144 andpre-miR451 are indicated with their corresponding expected sizes.Additional fragments released by possible Drosha processing of the 5′miR-451 flank are indicated with asterisks. Flanks are indicated witharrowheads. (FIG. 5B). Northern blots for confirmation of in-vitroDrosha processing assays. (FIG. 5C). Effect on mature miRNA levels ofDicer conditional ablation in Dicer flox/flox Cre-ER ES cells. (FIG. 5D)Effects on mature miRNA levels in Dicer null stable ES cells. (FIG. 5E).Mature miR-451 expression in dicer-null stable ES cells. U6 is used as aloading control. (FIG. 5F).

FIG. 6A-FIG. 6D show Ago2 catalysis is required for miR-451 biogenesis.Left panels: Northern blot on total RNA from wt and mutant liversprobing for miR-451 guide strand and passenger arms of the hairpin(indicated). Let-7 is used as a loading control. Right panels: Northernblots of Ago2 and IgG control immunoprecipitates from wt and mutantliver extracts with the indicated probes. (FIG. 6A). miRNA read lengthdistribution for the indicated miRNA from deep sequencing of WT andmutant livers. (FIG. 6B). Prediction of a miR-451 Ago2 cleavage site.top: miR-451 3′ end heterogeneity. Bottom: predicted cleavage site atthe 30th phosphodiester bond of pre-miR-451. (FIG. 6C). Left gel:in-vitro cleavage assay of pre-miR-451 by an Ago2 immunoprecipitate.Right gel: confirmation of the 3′ end character of the Ago2 cleavageproduct using beta elimination and 3′ end ligation reactions. (FIG. 6D).

FIG. 7 shows expression of miR-451 shRNA structural mimics. Amir-144-451 fragment cloned in the MluI/BglII site of vector andencompassing the mir-144-455 cluster sequence is amplified out of thehuman genome. From the amplified fragment, a miR-451 cassette isgenerated by subcloning a fragment of the mir-144-451 cluster sequencecomprising 5′ and 3′ miR-451 flanking sequences, engineering restrictionsites on each of the 5′ and 3′ ends of that fragment, and subcloning theresulting cassette into an MSCV expression plasmid backbone. An MSCVexpression construct encoding a miR-451 shRNA mimic targeted against p53mRNA is generated by replacing the native miR-451 precursor sequence(shaded portion; AAACCGTTACCATTACTGAGTTTAGTAATGGTAATGGTTCT) (SEQ ID NO:11) with a sequence encoding the mir-451 shRNA mimic

FIG. 8 shows that MicroRNA-451 based shRNA precursors (drosha products)are functional in mouse embryonic stem (ES) cells and manifest adifferent dose response compared to miR-30 based shRNAs precursormimics.

FIG. 9A-FIG. 9C shows that primary MicroRNA-451 based shRNA isfunctional, allowing the stable expression of the miR-451 mimics using amiR-451 backbone.

FIG. 10A-FIG. 10D show that primary Micro-451 based shRNAs are processedthrough the miR-451 pathway.

FIG. 11A-FIG. 11B shows the generation of a graftable primary miR-451longer backbone through the introduction of Restriction sites into theendogenous miR-451 sequence for cloning purposes. (FIG. 11A), Design ofrestriction sites in the miR-144-451 cloned backbone into MSCVretroviral expression vector. Note that restriction sites are locatedoutside of the predicted Drosha complex recognition region. Flankingsequences are in lower case with the extent of the drosha processedprecursor being covered by the gray bar. In the present case, the droshaprocessed product can include now 3′ flanking nucleotides. (FIG. 11B)miR-451 luciferase sensor assay analysis showing no interruption ofmature microRNA silencing efficiency. Titrations of MiR-451-fireflyluciferase sensor construct and endogenous primary microRNA constructsharboring wild-type or mutant miR-451 with the corresponding restrictionsites from (FIG. 11A) co-transfected with renilla constructs. Luciferaselevel was measured by dual luciferase reporter assay. MSCV-PIG is usedas a negative control.

FIG. 12 shows an example of miR-451 mimic design. Predicted secondarystructure of the minimal endogenous primary miR-451 sequence used forstable expression. The mimics are grafted within the shRNA variableregion highlighted in green according to the target sequence in thegene. Note that the GU wobble is converted to a perfect base pair in themimics, the bulge in the basal stem is conserved and we have shownexperimentally that the deletion of the T at position 41 makesnon-functional mimics, suggesting a structural requirement for droshaprocessing.

FIG. 13A-FIG. 13B show HOTAIR shRNA knockdown in stable MCG7 linesexpressing miR-451 mimic hairpins or miR30 mimic hairpins (FIG. 13A).Experiments were also conducted using siRNA HOTAIR knockdown in theMCF-7 cell line (FIG. 13B).

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 General Definitions

As used herein, the term “sequence” may mean either a strand or part ofa strand of nucleotides, or the order of nucleotides within a strand aor part of a strand, depending on the appropriate context in which theterm is used. Unless specified otherwise in context, the order ofnucleotides is recited from the 5′ to the 3′ direction of a strand.

A “coding sequence” or a sequence “encoding” a particular molecule is anucleic acid that is transcribed (in the case of DNA) or translated (inthe case of mRNA) into an inhibitory RNA (e.g., an shRNA or anantisense) or polypeptide, in vitro or in vivo, when operably linked toan appropriate regulatory sequence. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from prokaryotic or eukaryoticmRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, andsynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

As used herein, a sequence “directly following” a first sequence, indescribing an shRNA, means a sequence extending from, e.g. the 3′ endof, the first sequence without any nucleotides intervening therebetween.

As used herein, the term “fully complementary” with regard to a sequencerefers to a complement of the sequence by Watson-Crick base pairing,whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs witheither uracil (U) or thymine (T). A sequence may be fully complementaryto the entire length of another sequence, or it may be fullycomplementary to a specified portion or length of another sequence. Oneof skill in the art will recognize that U may be present in RNA, andthat T may be present in DNA. Therefore, an A within either of a RNA orDNA sequence may pair with a U in a RNA sequence or T in a DNA sequence.

As used herein, the term “wobble base pairing” with regard to twocomplementary nucleic acid sequences refers to the base pairing of G touracil U rather than C, when one or both of the nucleic acid strandscontains the ribonucleobase U.

As used herein, the term “substantially fully complementary” with regardto a sequence refers to the reverse complement of the sequence allowingfor both Watson-Crick base pairing and wobble base pairing, whereby Gpairs with either C or U, and A pairs with either U or T. A sequence maybe substantially complementary to the entire length of another sequence,or it may be substantially complementary to a specified portion orlength of another sequence. One of skill in the art will recognize thatthe U may be present in RNA, and that T may be present in DNA.Therefore, a U within an RNA sequence may pair with A or G in either anRNA sequence or a DNA sequence, while an A within either of a RNA or DNAsequence may pair with a U in a RNA sequence or T in a DNA sequence.

As used herein, the term “canonical” with regard to RNAi means,requiring cleavage by DICER for the maturation of an shRNA molecule.Therefore, a “canonical shRNA” is an shRNA that requires cleavage byDICER before becoming a mature shRNA, and a “canonical pathway” as itrelates to shRNA-mediated RNAi is a pathway involving the cleavage of anon-mature shRNA by DICER.

The term “gene” refers to a nucleic acid comprising an open readingframe encoding a polypeptide, including both exon and (optionally)intron sequences. The nucleic acid can also optionally includenon-coding sequences such as promoter and/or enhancer sequences.

As used herein, the term “long non-coding RNA” refers to a non-proteincoding RNA transcript longer than 200 nucleotides.

The term “mRNA” refers to a nucleic acid transcribed from a gene fromwhich a polypeptide is translated, and may include non-translatedregions such as a 5′UTR and/or a 3′UTR. It will be understood that anshRNA of the invention may comprise a nucleotide sequence that iscomplementary to any sequence of an mRNA molecule, including translatedregions, the 5′UTR, the 3′UTR, and sequences that include both atranslated region and a portion of either 5′UTR or 3′UTR. An shRNA ofthe invention may comprise a nucleotide sequence that is complementaryto a region of an mRNA molecule spanning the start codon or the stopcodon.

“Library” refers to a collection of nucleic acid molecules (circular orlinear). In one preferred embodiment, a library (alternatively referredto as a cDNA library) is representative of all expressed genes in acell, tissue, organ or organism. A library may also comprise randomsequences made by de novo synthesis, mutagenesis or other modificationor alteration of one or more sequences. A library may be contained inone or more vectors.

“Nucleic acid” refers to polynucleotides such as deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). The term can include single-strandedand double-stranded polynucleotides.

“Operably linked” means that the coding sequence is linked to aregulatory sequence in a manner which allows expression of the codingsequence. Regulatory sequences include promoters, enhancers, and otherexpression control elements that are art-recognized and are selected todirect expression of the coding sequence.

“Recombinant” RNA molecules are those produced by recombinant DNAtechniques; i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired RNA. “Synthetic” RNA molecules are thoseprepared by chemical synthesis.

A “subject” or “patient” can be a human or non-human animal.

A “transduced cell” is one that has been genetically modified. Geneticmodification can be stable or transient. Methods of transduction (i.e.,introducing vectors or constructs into cells) include, but are notlimited to, liposome fusion (transposomes), viral infection, and routinenucleic acid transfection methods such as electroporation, calciumphosphate precipitation and microinjection. Successful transduction willhave an intended effect in the transduced cell, such as gene expression,gene silencing, enhancing a gene target, or triggering targetphysiological event.

In one embodiment, “treating” means slowing, stopping or reversing theprogression of a disease or disorder. “Treating” can also meanamelioration of symptoms associated with a disease or disorder.

“Vector” refers to a vehicle for introducing a nucleic acid into a cell.Vectors include, but are not limited to, plasmids, phagemids, viruses,bacteria, and vehicles derived from viral or bacterial sources. Vectorscan also include aptamers, where the aptamer either forms part of, or isconjugated to the RNAi molecule (Dassie et al., Nature Biotechnology 27,839-846 (2009), Zhou and Rossi, Silence, 1:4 (2010), McNamera et al.,Nature Biotechnology 24, 1005-1015 (2006)). A “plasmid” is a circular,double-stranded DNA molecule. A useful type of vector for use in thepresent invention is a viral vector, wherein heterologous DNA sequencesare inserted into a viral genome that can be modified to delete one ormore viral genes or parts thereof. Certain vectors are capable ofautonomous replication in a host cell (e.g., vectors having an origin ofreplication that functions in the host cell). Other vectors can bestably integrated into the genome of a host cell, and are therebyreplicated along with the host genome.

4.2 RNAi Molecules

Interfering RNA or small inhibitory RNA (RNAi) molecules include shortinterfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), andmicro-RNAs (miRNAs) in all stages of processing, including shRNAs,pri-miRNAs, and pre-miRNAs. These molecules have different origins:siRNAs are processed from double-stranded precursors (dsRNAs) with twodistinct strands of base-paired RNA; siRNAs that are derived fromrepetitive sequences in the genome are called rasiRNAs; miRNAs arederived from a single transcript that forms base-paired hairpins. Basepairing of siRNAs and miRNAs can be perfect (i.e., fully complementary)or imperfect, including bulges in the duplex region.

The design of miR-451 shRNA mimics useful in this invention, and inparticular, the choice of target sequences for miR-451 shRNA mimics canbe based on existing shRNA, siRNA, piwi-interacting RNA (piRNA), microRNA (miRNA), double-stranded RNA (dsRNA), antisense RNA, or any otherRNA species that can be cleaved inside a cell to form interfering RNAs,with compatible modifications described herein. As used herein, shRNAsuseful in this invention include, without limitation, modified shRNAs,including shRNAs with enhanced stability in vivo. Modified shRNAsinclude molecules containing nucleotide analogues, including thosemolecules having additions, deletions, and/or substitutions in thenucleobase, sugar, or backbone; and molecules that are cross-linked orotherwise chemically modified. The modified nucleotide(s) may be withinportions of the shRNA molecule, or throughout it. For instance, theshRNA molecule may be modified, or contain modified nucleic acids inregions at its 5′ end, its 3′ end, or both, and/or within the guidestrand, passenger strand, or both, and/or within nucleotides thatoverhang the 5′ end, the 3′ end, or both. (See Crooke, U.S. Pat. Nos.6,107,094 and 5,898,031; Elmen et al., U.S. Publication Nos.2008/0249039 and 2007/0191294; Manoharan et al., U.S. Publication No.2008/0213891; MacLachlan et al., U.S. Publication No. 2007/0135372; andRana, U.S. Publication No. 2005/0020521; all of which are herebyincorporated by reference.)

As used herein, an “shRNA molecule” includes a conventional stem-loopshRNA, which forms a precursor miRNA (pre-miRNA). “shRNA” also includesmicro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strandand the passenger strand of the miRNA duplex are incorporated into anexisting (or natural) miRNA or into a modified or synthetic (designed)miRNA. When transcribed, a conventional shRNA (i.e., not a miR-451 shRNAmimic) forms a primary miRNA (pri-miRNA) or a structure very similar toa natural pri-miRNA. The pri-miRNA is subsequently processed by Droshaand its cofactors into pre-miRNA. Therefore, the term “shRNA” includespri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand or duplex (stem portion) that islinked on one side by a region of predominantly single-strandednucleotides (loop portion). The terms “hairpin” and “fold-back”structures are also used herein to refer to stem-loop structures. Suchstructures are well known in the art and the term is used consistentlywith its known meaning in the art. As is known in the art, the secondarystructure does not require exact base-pairing. Thus, the stem caninclude one or more base mismatches or bulges. Alternatively, thebase-pairing can be exact, i.e. not include any mismatches.

“RNAi-expressing construct” or “RNAi construct” is a generic term thatincludes nucleic acid preparations designed to achieve an RNAinterference effect. An RNAi-expressing construct comprises an RNAimolecule that can be cleaved in vivo to form an siRNA or a mature shRNA.For example, an RNAi construct is an expression vector capable of givingrise to an siRNA or a mature shRNA in vivo. Non-limiting examples ofvectors that may be used in accordance with the present invention aredescribed herein, for example, in section 4.6. Exemplary methods ofmaking and delivering long or short RNAi constructs can be found, forexample, in WO01/68836 and WO01/75164.

4.3 Use of RNAi

RNAi is a powerful tool for in vitro and in vivo studies of genefunction in mammalian cells and for therapy in both human and veterinarycontexts. Inhibition of a target gene is sequence-specific in that genesequences corresponding to a portion of the RNAi sequence, and thetarget gene itself, are specifically targeted for genetic inhibition.Three mechanisms of utilizing RNAi in mammalian cells have beendescribed. The first is cytoplasmic delivery of siRNA molecules, whichare either chemically synthesized or generated by DICER-digestion ofdsRNA. These siRNAs are introduced into cells using standardtransfection methods. The siRNAs enter the RISC to silence target mRNAexpression.

The second mechanism is nuclear delivery, via viral vectors, of geneexpression cassettes expressing a short hairpin RNA (shRNA). The shRNAis modeled on micro interfering RNA (miRNA), an endogenous trigger ofthe RNAi pathway (Lu et al., 2005, Advances in Genetics 54: 117-142,Fewell et al., 2006, Drug Discovery Today 11: 975-982). ConventionalshRNAs, which mimic pre-miRNA, are transcribed by RNA Polymerase II orIII as single-stranded molecules that form stem-loop structures. Onceproduced, they exit the nucleus, are cleaved by DICER, and enter theRISC as siRNAs.

The third mechanism is identical to the second mechanism, except thatthe shRNA is modeled on primary miRNA (shRNAmir), rather than pre-miRNAtranscripts (Fewell et al., 2006). An example is the miR-30 miRNAconstruct. The use of this transcript produces a more physiologicalshRNA that reduces toxic effects. The shRNAmir is first cleaved toproduce shRNA, and then cleaved again by DICER to produce siRNA. ThesiRNA is then incorporated into the RISC for target mRNA degradation.

For mRNA degradation, translational repression, or deadenylation, maturemiRNAs or siRNAs are loaded into the RNA Induced Silencing Complex(RISC) by the RISC-loading complex (RLC). Subsequently, the guide strandleads the RISC to cognate target mRNAs in a sequence-specific manner andthe Slicer component of RISC hydrolyses the phosphodiester boundcoupling the target mRNA nucleotides paired to nucleotide 10 and 11 ofthe RNA guide strand. Slicer forms together with distinct classes ofsmall RNAs the RNAi effector complex, which is the core of RISC.Therefore, the “guide strand” is that portion of the double-stranded RNAthat associates with RISC, as opposed to the “passenger strand,” whichis not associated with RISC.

4.4 Identification of an Alternative miRNA Biogenesis Pathway

Disclosed herein is that the erythroid specific miRNA miR-451, ischanneled through a novel small RNA biogenesis pathway requires AGO2catalysis and is independent of DICER processing. miR-451 is processedby Drosha, its maturation does not require Dicer. Instead, the pre-miRNAbecomes loaded into AGO2 and is cleaved by the AGO catalytic center togenerate an intermediate 3′ end, which is then further trimmed (Cheloufiet al., Nature 465(7298): 584-9 (2010)) (FIG. 3A-FIG. 3B).

4.5 Design of miR-451 shRNA Mimics

One can design and express miR-451 shRNA mimics based on the features ofthe native gene encoding the miR-451 shRNA. In particular, the miR-451architecture can be used to express miR-451 shRNA mimics from pol IIpromoter-based expression plasmids by using a variety of RNA polII-based expression vectors or even from pol III promoter-basedexpression plasmids using pol III-dependent promoters. In certainembodiments, expression vectors may employ use of expression cassettescomprising the miR-451 shRNA mimic. In certain embodiments, expressionvectors encoding miR-451 shRNA mimics may be based on CMV-based orMSCV-based vector backbones. In certain embodiments, expression vectorsencoding miR-451 shRNA mimics may be based on self-inactivatinglentivirus (SIN) vector backbones. Generally, appropriate vectorbackbones include vector backbones used in construction of expressionvectors for conventional shRNAs, including mir-30 based shRNAs.Exemplary use of expression cassettes in construction of shRNAexpression vectors also useful in the construction of expressioncassettes encoding the miR-451 shRNA mimics of the invention are foundin the following references: Gottwein E. and Cullen B. Meth. Enzymol.427:229-243, 2007, Dickens et al., Nature Genetics, 39:914-921, 2007,Chen et al., Science 303: 83-86, 2004; Zeng and Cullen, RNA 9: 112-123,2003, the contents of which are specifically incorporated herein byreference.

In certain embodiments, use the miR-451 shRNA mimics may employprecursor molecules comprised of flanking sequences. The precursormolecule is composed of any type of nucleic acid based molecule capableof accommodating such flanking sequences and the miR-451 shRNA mimicsequence. In certain embodiments, the methods for efficient expressionof the miR-451 shRNA mimics involve the use of expression vectorscomprising sequences encoding a precursor molecule, wherein the encodedprecursor molecule is a miR-451 shRNA mimic in the context of flankingsequences. In some embodiments, the flanking sequences comprise primarymiR-451 sequences. In some embodiments, the flanking sequences compriseprimary sequences from an miRNA or miRNAs other than miR-451. In someembodiments the primary miRNA sequences used as, or as part of theflanking sequences may direct drosha cleavage of the miRNA. In general,this type of approach in using precursor miRNAs and the individualcomponents of the precursor (flanking sequences and microRNA sequence)are provided in U.S. Publication No. 2008/0226553, which is specificallycited and incorporated by reference herein.

To investigate sequence versus structural requirements of miR-451 forentry into the alternative miRNA biogenesis pathway, we engineeredstructural mimics of miR-451 that might instead produce let-7c. At theconcentration tested, these structural mimics were as potent as thenative pre-let-7c in suppressing a GFP or luciferase reporter containingperfect let-7 complementary sites (FIG. 1A-C). The miR-451 precursorcould also be remodeled to express an shRNA that efficiently repressesp53 (FIG. 1D). We demonstrate that the primary transcript encoded from atransiently transfected miR-451 MSCV plasmid is processed to its matureform in human embryonic kidney 293 cells and only AGO2 is loaded withthe mature form of the molecule (FIG. 2A-FIG. 2B). The miR-451expression plasmid is also processed in mouse embryonic fibroblast andmouse embryonic stems cells (Cheloufi et al., Nature 465(7298): 584-9(2010)).

These results demonstrate that by engineering shRNA molecules that mimicthe structure of miR-451, processing of these shRNA in mammalian cellsis channeled into the alternative miRNA biogenesis pathway. In oneaspect of the invention, miR-451 shRNA mimics may be used forsuppression or silencing of particular expressed genes through apost-transcriptional mechanism by targeting the shRNA against theexpressed mRNA. In another aspect of the invention, miR-451 mimics maybe used for suppression or silencing of particular expressed genesthrough a transcriptional mechanism, by targeting the shRNA againstintrons or other non-coding regions of the target gene.

In one aspect of the invention, design of a miR-451 shRNA mimic may bebased on the sequence of an siRNA targeted against the target gene. Inanother aspect, design of an miR-451 shRNA mimic may be based on any21-23 nt sequence in the coding sequence of a target gene. In anotheraspect, design of a miR-451 shRNA mimic may be based on any 21-23 ntsequence in an intron or other non-coding region of a target gene. Inparticular, the miR-451 shRNA mimic comprises a sequence that is fullycomplementary to a 21 to 23 long nucleotide sequence in the target gene,or to the 21 to 23 nucleotide target sequence of the siRNA. In designingthe miR-451 shRNA mimic, this fully complementary sequence is positionedwithin the shRNA, such that Ago2 processing of the shRNA and furthertrimming within the RISC complex generates an active silencing moleculecomprising said fully complementary sequence.

In a non-limiting example, design of a miR-451 mimic shRNA targeting p53is depicted in (FIG. 3-FIG. 3B). The resulting ˜40 nt shRNA has a shortstem and a tight loop and cannot be processed by DICER. Instead, it iscleaved by AGO2 and then further trimmed to generate the active strands.

In some embodiments of the invention, an shRNA comprises a firstsequence of 21, 22, or 23 nucleotides fully complementary to a sequencein a non-coding target gene.

In some embodiments of the invention, an shRNA comprises a firstsequence of 21, 22, or 23 nucleotides fully complementary to a sequencein a target long non-coding RNA.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence is fullycomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence.

In some embodiments, the second sequence is at least 60% complementaryto the first 17 or 18 nucleotides counted from the 5′ end of the firstsequence. The second sequence may be at least 60% complementary to thefirst 17 or 18 nucleotides counted from the 5′ end of the first sequencealong its entire length, or along a portion of the first 17 or 18nucleotides counted from the 5′ end of the first sequence, provided thesecond sequence is capable of hybridizing with the first sequence undernormal physiological conditions. In some embodiments, the secondsequence may be from 60 to 99% complementary to the first 17 or 18nucleotides counted from the 5′ end of the first sequence. The secondsequence may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% complementarycomplementary to the first 17 or 18 nucleotides counted from the 5′ endof the first sequence.

In some embodiments, an shRNA comprises a first sequence of 21, 22 or 23nucleotides which is complementary to a sequence in a target mRNAmolecule, gene, or long non-coding RNA and a second sequence directlyfollowing the first sequence, wherein the second sequence iscomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence. In some embodiments the firstsequence is at least 60% complementary to a sequence in a target mRNAmolecule, gene, or long non-coding RNA. The first sequence may be atleast 60% complementary to a sequence in a target mRNA molecule or genealong its entire length, or along portions of its length, provided atleast 12 nucleotides are complementary between the two sequences,continuously or non-continuously, and provided the first sequence iscapable of hybridizing with the sequence in the target mRNA molecule,gene, or long non-coding RNA under normal physiological conditions. Insome embodiments, the first sequence may be from 60 to 99% complementaryto a sequence in a target mRNA molecule, gene, or long non-coding RNA.The first sequence may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%complementary to a sequence in a target mRNA molecule, gene, or longnon-coding RNA.

In some embodiments, an shRNA of the invention may be an isolated shRNA.

In various embodiments of the instant shRNA, the last 3 nucleotides, oralternatively the last 4 nucleotides, of the first sequence form a loopregion in the short hairpin molecule.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 3′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 3′ end.

In various embodiments of the instant shRNA, the shRNA has a 1nucleotide overhang at its 5′ end, or alternatively a 2, 3 or more than3 nucleotide overhang at its 5′ end.

In an embodiment of the instant shRNA, the shRNA has no 3′ or 5′overhang.

In an embodiment of the instant shRNA, the shRNA consists of a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence is fullycomplementary to the sequence of the first 17 or 18 nucleotides countedfrom the 5′ end of the first sequence.

Another aspect of the invention provides for design of non-naturallyoccurring miRNAs based on structural mimics of miR-451 precursors. Incertain embodiments, such structural miRNA mimics of miR-451, or anexpression vector or library of expression vectors encoding suchstructural mimics can be introduced into different genetic backgroundsof mammalian cells, in particular in cells deficient of the canonicalpathway enzymes, such as dicer, to screen for and identify such miRNAscapable of rescuing the null phenotype and the functional roles of suchmiRNAs in contributing to the phenotype.

Another aspect of the invention provides for the design of non-naturallyoccurring miRNA mimicks of miR-451 precursors that comprise the guidesequence of a naturally occurring canonical shRNA. Certain embodimentsof the invention comprise an expression vector or libraries ofexpression vectors encoding such shRNAs. In some embodiments, an shRNAof the invention may be designed to target a sequence normally targetedby a canonical miRNA. In some embodiments, the shRNA, or a library ofshRNAs may be expressed in cells deficient in one or more of thecanonical pathway enzymes such as dicer, to screen for and/or identifysuch miRNAs capable of rescuing the null phenotype, or part of the nullphenotype, and/or the functional roles of such miRNAs in contributing toa phenotype or a part of a phenotyope. One of skill in the art willunderstand that miRNAs are not always fully complementary to theirtarget sequences, including those in the 3′UTR of a target mRNA. In someembodiments, non-naturally occurring miRNA mimicks of miR-451 precursorscomprise the guide sequence of a canonical shRNA that is between 60 and100% complementary to one or more target sequences. In some embodimentsa guide sequence may be 60, 65, 75, 80, 85, 90, 95, or 99% complementaryto its target sequence(s). One of skill in the art will understand thata guide sequence may have multiple target sequences for which it mighthave differing complementarity.

In one aspect of the invention, a non-naturally occurring miRNA isprovided comprising a first sequence of 21, 22 or 23 nucleotidescorresponding to the entire mature sequence, or a portion of thatsequence, of a mammalian miRNA other than miR-451, and a second sequencedirectly following the first sequence, wherein the second sequence isfully complementary to the sequence of the first 17 or 18 nucleotidescounted from the 5′ end of the first sequence. The skilled practitionerwill appreciate that the mature sequence of a mammalian miRNAalternatively may be identified as the sequence of the guide strand, orguide sequence for that miRNA.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 19, 20 or 21 nucleotides fully complementary to a sequencein a target gene, having a sequence other than the mature sequence ofmiR-451, and a second sequence directly following the first sequence,wherein the second sequence is fully complementary to the sequence ofthe first 15 or 16 nucleotides counted from the 5′ end of the firstsequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides complementary to a sequence in atarget gene, and a second sequence directly following the firstsequence, wherein the second sequence is fully complementary to thesequence of the first 17 nucleotides counted from the 5′ end of thefirst sequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides fully complementary to a sequencein the coding region of a target gene, and a second sequence directlyfollowing the first sequence, wherein the second sequence iscomplementary to the sequence of the first 17 nucleotides counted fromthe 5′ end of the first sequence.

In one aspect of the invention, an shRNA is provided comprising a firstsequence of 21, 22 or 23 nucleotides complementary to a sequence in atarget gene, having a sequence other than the mature sequence ofmiR-451, and a second sequence directly following the first sequence,wherein the second sequence is complementary to the sequence of thefirst 17 nucleotides counted from the 5′ end of the first sequence.

In some embodiments, the first sequence of 21, 22 or 23 nucleotides isfully complementary to a sequence in a target gene.

In some embodiments, the first sequence of 21, 22 or 23 nucleotides iscomplementary to a coding region of the target gene.

In some embodiments, the first sequence of 21, 22 or 23 nucleotides iscomplementary to a sequence in an mRNA molecule encoded by the gene,wherein the sequence in the mRNA molecule is present in the sequence ofthe target gene.

In some embodiments, the first sequence of 21, 22 or 23 nucleotides iscomplementary to a 3′ untranslated region (UTR) sequence in an mRNAmolecule encoded by the gene, wherein the 3′ UTR sequence in the mRNAmolecule is present in the sequence of the target gene.

In some embodiments, the second sequence directly following the firstsequence is fully complementary to the sequence of the first 18nucleotides counted from the 5′ end of the first sequence. In someembodiments, the shRNA consists of from 38 to 50 nucleotides.

In one aspect of the invention, an shRNA is provided having thestructure

wherein X₂ to X₂₂ are nucleotides complementary to a sequence in atarget gene, and are in a sequence other than the mature sequence ofmiR451; Y₄ to Y₂₀ are nucleotides complementary to X₂ to X₁₈; and X₁,Y₁, Y₂, and Y₃, are nucleotides that may be present or absent, wherein,X₁ and Y₃, when present, may be complementary or not complementary

In various embodiments of the instant non-naturally occurring miRNA, thelast 3 nucleotides, or alternatively the last 4 nucleotides, of thefirst sequence form a loop region in the short hairpin molecule.

In various embodiments of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has a 1 nucleotide overhang at its 3′ end,or alternatively a 2, 3 or more than 3 nucleotide overhang at its 3′end.

In various embodiments of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has a 1 nucleotide overhang at its 5′ end,or alternatively a 2, 3 or more than 3 nucleotide overhang at its 5′end.

In an embodiment of the instant non-naturally occurring miRNA, thenon-naturally occurring miRNA has no 3′ or 5′ overhang.

In some embodiments, the shRNA of the invention is a synthetic shRNA.

As non-limiting examples, in certain embodiments the instantnon-naturally occurring miRNA can comprise a sequence, selected from themature sequence of human miR-18b (uaaggugcaucuagugcaguuag) (SEQ ID NO:12) of 21, 22 or 23 nucleotides: for example, uaaggugcaucuagugcaguu (SEQID NO: 13), uaaggugcaucuagugcaguua (SEQ ID NO: 14),uaaggugcaucuagugcaguuag (SEQ ID NO: 15), aaggugcaucuagugcaguuag (SEQ IDNO: 16), aggugcaucuagugcaguuag (SEQ ID NO: 17). In certain embodimentsthe instant non-naturally occurring miRNA can comprise a sequence,selected from the mature sequence of humanmiR-21(uagcuuaucagacugauguuga) (SEQ ID NO: 18) of 21 or 22 nucleotides:for example, uagcuuaucagacugauguug (SEQ ID NO: 19),uagcuuaucagacugauguuga (SEQ ID NO: 20), agcuuaucagacugauguuga (SEQ IDNO: 21). In other embodiments, non-naturally occurring miRNA cancomprise a sequence of 21, 22 or 23 nucleotides selected from the maturesequence of any other human or mammalian miRNA, wherein such sequencesare readily available through public databases, such as miRBase,(Griffiths-Jones et al., miRBase: tools for microRNA genomics, NAR,2008, Vol. 36, Database issue D154-D158), accessible athttp://www.mirbase.org/, such mature miRNA sequences available onmiRBase specifically incorporated herein by reference.

In certain preferred embodiments, the instant non-naturally occurringmiRNA can comprise a sequence, selected from the mature sequence of ahuman or mammalian miRNA with known involvment in cancer and otherdiseases, or those with known involvement in development anddifferentiation, for example such miRNAs, and mature sequences thereof,identified in United States Patents and Patent Publications U.S. Pat.Nos. 7,232,806, 7,307,067, 7,670,840, US 2010/0048674 (Feb. 25, 2010),US 2007/0072204 (Mar. 29, 2007), US 2009/0124566 (May 14, 2009), US2009/0176723 (Jul. 9, 2009), US 2009/0186353 (Jul. 23, 2009) and US2009/0286242 (Nov. 19, 2009), the contents of which are hereinspecifically incorporated by reference.

In certain embodiments, a non-naturally occurring miRNA is providedcomprising a first sequence of 21, 22 or 23 nucleotides corresponding tothe entire sequence of the passenger strand of a mammalian miRNA, or aportion of that sequence, and a second sequence directly following thefirst sequence, wherein the second sequence is fully complementary tothe sequence of the first 17 or 18 nucleotides counted from the 5′ endof the first sequence. In certain embodiments, the instant non-naturallyoccurring miRNA can comprise a sequence of 21, 22 or 23 nucleotidesselected from the sequence of the passenger strand of any human ormammalian miRNA, wherein such passenger strand sequences, or annotationsdefining such sequences, are readily available through public databases,such as miRBase, (Griffiths-Jones et al., miRBase: tools for microRNAgenomics, NAR, 2008, Vol. 36, Database issue D154-D158), accessible athttp://www.mirbase.org/, wherein such sequences or annotationsidentifying such sequences, and available on miRBase, are specificallyincorporated herein by reference. Also included in the invention is anisolated non-mammalian cell comprising the shRNAs described herein. Thecells may be those of vertebrate organisms, or non-vertebrate organismssuch as insects. The cells may be those of fish (e.g. those of the Fugugenus or the Danio genus), frogs (e.g. those of the Xenopus genus),round worms (e.g. those of the Caenorhabdis genus), flies (such as theDrosophila genus), or others. Another aspect of the invention provides anon-human animal comprising the cell described above. In certainembodiments, the non-human animal may be a chimeric animal, some ofwhose somatic or germ cells comprising the shRNAs described herein.Alternatively, the non-human animal may be a transgenic animal, all ofwhose somatic or germ cells comprise the shRNAs described herein. Thus,transgenic animals whose genomes comprise a sequence encoding the shRNAsof the invention are also provided.

4.6 Vectors

In certain embodiments, expression vectors encoding miR-451 shRNA mimicsmay be based on CMV-based or MSCV-based vector backbones. In certainembodiments, expression vectors encoding miR-451 shRNA mimics may bebased on self-inactivating lentivirus (SIN) vector backbones. Generally,appropriate vector backbones include vector backbones used inconstruction of expression vectors for conventional shRNAs, includingmir-30 based shRNAs. Exemplary vector backbones and methodologies forconstruction of expression vectors suitable for use with the miR-451shRNA mimics of this invention, and methods for introducing suchexpression vectors into various mammalian cells are found in thefollowing references: Premsrurit P K. et al., Cell, 145(1):145-158,2011, Gottwein E. and Cullen B. Meth. Enzymol. 427:229-243, 2007,Dickens et al., Nature Genetics, 39:914-921, 2007, Chen et al., Science303: 83-86, 2004; Zeng and Cullen, RNA 9: 112-123, 2003, the contents ofwhich are specifically incorporated herein by reference.

The vectors can be targeting vectors, such as those using flprecombination into the colA locus allowing single copy integration.Other targeting sites in the mouse genome include but are not limited toROSA26 and HPRT. Additionally, transposase may be used to introducemimics into the genome of an animal or the cell of an animal. See,Premsrurit P K. et al., Cell, 145(1):145-158, (2011), the contents ofwhich are specifically incorporated herein by reference.

The vectors described in International application no. PCT/US2008/081193(WO 09/055724) and methods of making and using the vectors areincorporated herein by reference. The disclosure provided thereinillustrates the general principles of vector construction and expressionof sequences from vector constructs, and is not meant to limit thepresent invention.

shRNAs can be expressed from vectors to provide sustained silencing andhigh yield delivery into almost any cell type. In a certain embodiment,the vector is a viral vector. Exemplary viral vectors includeretroviral, including lentiviral, adenoviral, baculoviral and avianviral vectors. The use of viral vector-based RNAi delivery not onlyallows for stable single-copy genomic integrations but also avoids thenon-sequence specific response via cell-surface toll-like receptor 3(TLR3), which has raised many concerns for the specificity of siRNAmediated effects. In one embodiment of the present invention, a pool ofshRNAs is introduced into murine HSCs using a vector known in the art.

Retroviruses from which the retroviral plasmid vectors can be derivedinclude, but are not limited to, Moloney Murine Leukemia Virus, spleennecrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosisvirus, gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviralplasmid vector can be employed to transduce packaging cell lines to formproducer cell lines. Examples of packaging cells which can betransfected include, but are not limited to, the PE50l, PA3l7, R-2,R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectorcan transduce the packaging cells through any means known in the art. Aproducer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding a DNA replication protein. Suchretroviral vector particles then can be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express a DNA replication protein.

In certain embodiments, cells can be engineered using anadeno-associated virus (AAV). AAVs are naturally occurring defectiveviruses that require helper viruses to produce infectious particles(Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It isalso one of the few viruses that can integrate its DNA into nondividingcells. Vectors containing as little as 300 base pairs of AAV can bepackaged and can integrate, but space for exogenous DNA is limited toabout 4.5 kb. Methods for producing and using such AAVs are known in theart. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,5,436,146, 5,474,935, 5,478,745, and 5,589,377. For example, an AAVvector can include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The recombinant AAV vector canbe transfected into packaging cells which are infected with a helpervirus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe polynucleotide construct. These viral particles are then used totransduce eukaryotic cells.

In certain embodiments, cells can be engineered using a lentivirus andlentivirus based vectors. Such an approach is advantageous in that itallows for tissue-specific expression in animals through use of celltype-specific pol II promoters, efficient transduction of a broad rangeof cell types, including nondividing cells and cells that are hard toinfect by retroviruses, and inducible and reversible gene knockdown byuse of tet-responsive and other inducible promoters. Methods forexpressing shRNAs by producing and using lentivirus engineered cells areknown in the art. For exemplary descriptions of such methods, see forexample, Stegmeier F. et al., Proc Natl Acad Sci USA 2005,102(37):13212-13217, Klinghoffer et al., RNA 2010, 16:879-884, thecontents of which are specifically incorporated herein. Efficientproduction of replication-incompetent recombinant lentivirus may beachieved, for example, by co-tranfection of expression vectors andpackaging plasmids using commercially available packaging cell lines,such as TLA-HEK293™, and packaging plasmids, available from ThermoScientific/Open Biosystems, Huntsville, Ala.

Essentially any method for introducing a nucleic acid construct intocells can be employed. Physical methods of introducing nucleic acidsinclude injection of a solution containing the construct, bombardment byparticles covered by the construct, soaking a cell, tissue sample ororganism in a solution of the nucleic acid, or electroporation of cellmembranes in the presence of the construct. A viral construct packagedinto a viral particle can be used to accomplish both efficientintroduction of an expression construct into the cell and transcriptionof the encoded shRNA. Other methods known in the art for introducingnucleic acids to cells can be used, such as lipid-mediated carriertransport, chemical mediated transport, such as calcium phosphate, andthe like. Thus the shRNA-encoding nucleic acid construct can beintroduced along with components that perform one or more of thefollowing activities: enhance RNA uptake by the cell, promote annealingof the duplex strands, stabilize the annealed strands, or otherwiseincrease inhibition of the target gene.

Expression of endogenous miRNAs is controlled by RNA polymerase II (PolII) promoters. It has been shown that shRNAs are also most efficientlydriven by Pol II promoters, as compared to RNA polymerase III promoters(Dickins et al., 2005, Nat. Genet. 39: 914-921). Therefore, in a certainembodiment, the coding sequence of the RNAi molecule is controlled by aninducible promoter or a conditional expression system, including,without limitation, RNA polymerase type II promoters. Examples of usefulpromoters in the context of the invention are tetracycline-induciblepromoters (including TRE-tight), IPTG-inducible promoters, tetracyclinetransactivator systems, and reverse tetracycline transactivator (rtTA)systems. Constitutive promoters can also be used, as can cell- ortissue-specific promoters. Many promoters will be ubiquitous, such thatthey are expressed in all cell and tissue types. A certain embodimentuses tetracycline-responsive promoters, one of the most effectiveconditional gene expression systems in in vitro and in vivo studies. SeeInternational Patent Application PCT/US2003/030901 (Publication No. WO2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11:975-982, for an exemplary description of inducible shRNA.

To facilitate the monitoring of the target gene knockdown, cellsharboring the RNAi-expressing construct can additionally comprise amarker or reporter construct, such as a fluorescent construct. Thereporter construct can express a marker, such as green fluorescentprotein (GFP), enhanced green fluorescent protein (EGFP), RenillaReniformis green fluorescent protein, GFPmut2, GFPuv4, yellowfluorescent protein (YFP), such as VENUS, enhanced yellow fluorescentprotein (EYFP), cyan fluorescent protein (CFP), enhanced cyanfluorescent protein (ECFP), blue fluorescent protein (BFP), enhancedblue fluorescent protein (EBFP), citrine and red fluorescent proteinfrom discosoma (dsRED). Other suitable detectable markers includechloramphenicol acetyltransferase (CAT), luminescent proteins such asluciferase lacZ (β-galactosidase) and horseradish peroxidase (HRP),nopaline synthase (NOS), octopine synthase (OCS), and alkalinephosphatase. The marker gene can be separately introduced into the cellharboring the shRNA construct (e.g., co-transfected, etc.).Alternatively, the marker gene can be on the shRNA construct, and themarker gene expression can be controlled by the same or a separatetranslation unit, for example, by an IRES (internal ribosomal entrysite). In one aspect of the invention, marker genes can be incorporatedinto “sensor” expression vectors for use in high throughput methods fordetermining the knockdown efficiency of miR-451 shRNA mimics targetedagainst particular genes and for identifying the most potent targetsequences for a particular target gene. Such methods, including thedesign and use of plasmids and reporter constructs for testing thepotency of particular shRNA molecules, here useful for testing thepotency of the miR-451 shRNA mimics are described in PCT publicationFellman et al., WO/2009/055724, the contents of which is hereinspecifically incorporated by reference in its entirety.

Reporters can also be those that confer resistance to a drug, such asneomycin, ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, doxycycline, and tetracyclin. Reporters can also be lethalgenes, such as herpes simplex virus-thymidine kinase (HSV-TK) sequences,as well as sequences encoding various toxins including the diphtheriatoxin, the tetanus toxin, the cholera toxin and the pertussis toxin. Afurther negative selection marker is the hypoxanthine-guaninephosphoribosyl transferase (HPRT) gene for negative selection in6-thioguanine.

To facilitate the quantification of specific shRNAs in a complexpopulation of cells infected with a library of shRNAs, each shRNAconstruct can additionally comprise a barcode. A barcode is a uniquenucleotide sequence (generally a 19-mer), linked to each shRNA. Thebarcode can be used to monitor the abundance of each shRNA via micoarrayhybridization (Fewell et al., 2006, Drug Discovery Today 11: 975-982).In a certain embodiment, each shRNA construct also comprises a uniquebarcode. For more information on the use of barcodes in shRNA pooledanalyses, see WO 04/029219, Bernards et al., 2006, Nature Methods 3:701-706, and Chang et al., 2006, Nature Methods 3: 707-714.

4.7 Methods of Treatment

In certain embodiments, the invention provides a composition formulatedfor administration of miR-451 shRNA mimics in vivo to a subject, such asa human or veterinary subject. A composition so formulated can comprisea stem cell comprising a nucleic acid construct encoding a miR-451 shRNAmimic designed to decrease the expression of a target gene. Acomposition can also comprise a pharmaceutically acceptable excipient.

For example, the miR-451 shRNA mimic can be reliably expressed in vivoin a variety of cell types. In certain embodiments the cells areadministered in order to treat a condition. There are a variety ofmechanisms by which shRNA expressing cells can be useful for treatingcancer and other diseases. For example, a condition can be caused, inpart, by a population of cells expressing an undesirable gene. Thesecells can be ablated and replaced with administered cells comprisingshRNA that decreases expression of the undesirable gene. An shRNA can betargeted to essentially any gene, the decreased expression of which canbe helpful in treating cancer or another disease.

Any suitable cell can be used. For example, cells to be transfected canbe essentially any type of cell for implantation into in a subject. Thecell having the target gene can be germ line or somatic, totipotent orpluripotent, dividing or non-dividing, parenchymal or epithelial,immortalized or transformed, or the like. The cell can be a stem cell ora differentiated cell. After transfection, stem cells can beadministered to a subject, or cultured to form differentiated stem cells(e.g., embryonic stem cells cultured to form neural, hematopoietic orpancreatic stem cells) or cultured to form differentiated cells.

Stem cells can be stem cells recently obtained from a donor, and incertain embodiments, the stem cells are autologous stem cells. Stemcells can also be from an established stem cell line that is propagatedin vitro. Suitable stem cells include embryonic stems and adult stemcells, whether totipotent, pluripotent, multipotent or of lesserdevelopmental capacity. Stem cells can be derived from mammals, such asrodents (e.g. mouse or rat), primates (e.g. monkeys, chimpanzees orhumans), pigs, or ruminants (e.g. cows, sheep and goats). Examples ofmouse embryonic stem cells include: the JM1 ES cell line described in M.Qiu et al., Genes Dev 9, 2523 (1995), and the ROSA line described in G.Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cellsdescribed in U.S. Pat. No. 6,190,910. Many other mouse ES lines areavailable from Jackson Laboratories (Bar Harbor, Me.). Examples of humanembryonic stem cells include those available through the followingsuppliers: Arcos Bioscience, Inc., Foster City, Calif.; CyThera, Inc.,San Diego, Calif.; ES Cell International, Melbourne, Australia; GeronCorporation, Menlo Park, Calif.; University of California, SanFrancisco, Calif.; and Wisconsin Alumni Research Foundation, Madison,Wis. In addition, examples of embryonic stem cells are described in thefollowing U.S. patents and published patent applications: U.S. Pat. Nos.6,245,566; 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780;20020045259; 20020068045. Examples of human adult stem cells includethose described in the following U.S. patents and patent applications:U.S. Pat. Nos. 5,486,359; 5,766,948; 5,789,246; 5,914,108; 5,928,947;5,958,767; 5,968,829; 6,129,911; 6,184,035; 6,242,252; 6,265,175;6,387,367; 20020016002; 20020076400; 20020098584; and, for example, inPCT publication WO 01/11011. In certain embodiments, a suitable stemcell can be derived from a cell fusion or dedifferentiation process,such as described in U.S. patent application 20020090722, in PCTpublications WO 02/38741, WO 01/51611, WO 99/63061, and WO 96/07732.

Transfected cells can also be used in the manufacture of a medicamentfor the treatment of subjects. Examples of pharmaceutically acceptableexcipients include matrices, scaffolds, or other substrates to whichcells can attach (optionally formed as solid or hollow beads, tubes, ormembranes), as well as reagents that are useful in facilitatingadministration (e.g. buffers and salts), preserving the cells (e.g.chelators such as sorbates, EDTA, EGTA, or quaternary amines or otherantibiotics), or promoting engraftment. Cells can be encapsulated in amembrane or in a microcapsule. Cells can be placed in microcapsulescomposed of alginate or polyacrylates. (Sugamori et at (1989) Trans. Am.Soc. Artif. Intern. Organs 35:791; Levesque et al. (1992) Endocrinology130:644; and Lim et al. (1992) Transplantation 53:1180).

Additional methods for encapsulating cells are known in the art.(Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat.No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaegeret al. (1990) Prog. Brain Res. 82:4146; and Aebischer et al. (1991) J.Biomech. Eng. 113:178-183, U.S. Pat. No. 4,391,909; U.S. Pat. No.4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; andAebischer et al. (1991) Biomaterials 12:50-55).

The site of implantation of cell compositions can be selected by one ofskill in the art depending on the type of cell and the therapeuticobjective. Exemplary implantation sites include intravenous orintraarterial administration, administration to the liver (via portalvein injection), the peritoneal cavity, the kidney capsule or the bonemarrow.

In certain embodiments, the invention provides for modification and invivo delivery of miR-451 shRNA mimics as synthetic RNAi molecules.Modification and in vivo delivery of synthetic RNAi molecules, includingshRNAs incorporating modified nucleotides, such as those with chemicalmodifications to the 2′—OH group in the ribose sugar backbone, such as2′-O-methyl (2′OMe), 2′-fluoro (2′F) substitutions, and those containing2′OMe, or 2′F, or 2′-deoxy, or “locked nucleic acid” (LNA) modificationscan be accomplished as described in U.S. Pat. Nos. 6,627,616, 6,897,068,6,379,966; in U.S. Patent Application Publication Nos. US. 2005/0107325(May 19, 2005), US 2007/0281900 (Dec. 6, 2007) and US 2007/0293449 (Dec.20, 2007); and in Vorhies and Nemunaitis J J, Methods Mol Biol. 2009;480:11-29, Lopez-Fraga M et al., Infect Disord Drug Targets. 2008December; 8(4):262-73, Watts et al., Drug Discov Today. 2008 October;13(19-20):842-55, Lu and Woodle, Methods Mol Biol. 2008; 437:93-107, deFougerolles et al., Hum Gene Ther. 2008 February; 19(2):125-32, Rossi JJ, Hum Gene Ther. 2008 April; 19(4):313-7, Belting M and Wittrup A.Methods Mol Biol. 2009; 480:1-10, Pushparaj et al., J. Dent. Res. 2008;87: 992-1003, Shrivastava and Srivastava, Biotechnol J. 2008 March;3(3):339-53, and Raemdonck K. et al., Drug Discov Today. 2008 November;13(21-22):917-31, CastanottoD & Rossi J J, Nature 2009 January;457:426-433, Davis M et al., Nature advance online publication (21 Mar.2010) doi:10.1038/nature08956, each of which are incorporated byreference in their entireties.

4.8 Screening Methods

Constructs encoding miR-451 shRNA mimics or libraries of such constructscan be introduced into intact cells/organisms and can be used inscreening, such as high throughput screening (HTS). For example, byusing miR-451 shRNA mimics or libraries of such mimics to knockdownexpression of target genes, the function of those target genes, forexample in disease, can be assessed. Similarly, potential targets forpharmaceuticals can be identified or studied using such methods. A panelof miR-451 shRNA mimics that affect target gene expression by varyingdegrees may be used. In particular, it may be useful to measure anycorrelation between the degree of gene expression decrease and aparticular phenotype.

Libraries of miR-451 shRNA mimics can be produced using methods known inthe art. For example, libraries of miR-451 shRNA mimics can based onexisting libraries, such as existing shRNA libraries. Existing materialsand methods for design and construction of expression cassettes,selection and modification of vectors and vector backbones, libraryconstruction, design of target sequences, and library validation, asapplied to conventional shRNA libraries may be applied in theconstruction of libraries comprised of the miR-451 shRNA mimics of thepresent invention. As non-limiting examples, such materials and methodsare described in Chang et al., Nature Meth. 3:707-714 (2006), PCTpublication Fellman et al., WO/2009/055724, the contents of which arespecifically incorporated herein by reference.

In certain aspects, the invention provides methods forscreening/evaluating gene function in vivo. A cell containing aconstruct for expression of a miR-451 shRNA mimic may be introduced intoan animal and a phenotype may be assessed to determine the effect of thedecreased gene expression. An entire animal may be generated from suchcells (e.g., ES cells) containing the miR-451 shRNA mimic construct. Aphenotype of the animal may be assessed. The animal may be essentiallyany experimentally tractable animal such as a non-human primate, arodent, a canine, a feline, etc. Populations of animals expressingdifferent members of a library of miR-451 shRNA mimics may also begenerated. The phenotypes of such animals may be assessed to determine,for example, the effect of a target gene on a disease phenotype (e.g.tumor initiation or growth), stem cell differentiation, drug sensitivity(e.g. sensitivity of tumor cells to chemotherapeutic drugs),susceptibility to a viral, bacterial or other infections, or any otherphenotype of interest, including disease phenotypes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

5. EXAMPLES OF THE INVENTION

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

5.1 Example 1: Identification of a Non-Canonical microRNAs BiogenesisPathway 5.1.1 Mature miR-451 Expression Depends on Ago2 Catalysis

To investigate the evolutionary pressure to conserve Argonaute enzymaticactivity, we engineered a mouse with catalytically inactive Ago2alleles. Homozygous mutants died shortly after birth with an obviousanemia. (Cheloufi et al., Nature 465(7298): 584-9 (2010)). Our resultssuggested that miRNA directed target cleavage might prove important forerythrocyte maturation. As a step toward identifying such a target, weprofiled miRNAs expressed in the liver, one of the fetal hematopoieticsites. Deep sequencing from wild-type animals and Ago2^(ADH) homozygotesrevealed that virtually all microRNAs were present at nearly identicallevels. However, one miRNA, miR-451, represented 11% of all miRNA readsin normal fetal liver but was dramatically reduced in the mutants (FIG.4A-FIG. 4C).

Previous studies have demonstrated a strong dependency of thedevelopment of pro-E into basophilic erythroblasts on the expression ofmiR-45137. Together, miR-451 and miR-144 form a microRNA cluster withrobust expression in erythroid cells. This pattern can be explained inpart based upon the presence of regulatory sites for the GATA-1 zincfinger transcription factor, which acts as a master regulator ofeythroid differentiation38. The regulatory circuit seems to be intact inAgo2ADH animals, since we observe no changes in the levels ofpri-mir144-451 in homozygous mutants (FIG. 4B). This strongly pointed toan impact of catalysis on miR-451 maturation rather than miR-451expression.

MicroRNA biogenesis occurs via a two-step processing pathway whereinDrosha initially cleaves the primary microRNA transcript to liberate ahairpin pre-miRNA 39. This is exported to the cytoplasm and recognizedand cleaved by Dicer to yield the mature duplex, which is loaded intoAgo. The passenger strand is removed through unknown mechanisms to yielda complex ready for target recognition.

An examination of the miR-451 precursor and its mature strand revealedan unusual feature. As annotated, the 6 terminal nucleotides of the 23nt long mature miR-451 span the loop region and extend into thecomplementary strand of the hairpin precursor. This arrangement appearsincompatible with the well-studied enzymatic activities of Drosha andDicer, which would normally liberate the mature microRNA mapping to thestem only (FIG. 4C). We therefore explored the possibility that miR-451might adopt an unusual mode of biogenesis.

5.1.3 Non-Canonical Biogenesis of miR-451

We began by assessing the dependency of miR-451 on Drosha. We created aconstruct, which drives the expression of the miR-144/451 precursor froma strong viral promoter and introduced this into MEF homozygous for aconditional Drosha allele. Following activation of Cre-ER and Droshaloss of function, we noted a 20-fold reduction in levels of maturemiR-451. This was even more dramatic than the effect on a miRNA, let-7c,with a well-established dependency on canonical processing factors (FIG.5A). We also assessed the ability of Drosha to liberate pre-miR-451 invitro. Drosha complexes were affinity purified from human 293T cells andmixed with in vitro synthesized fragments of pri-miR-451 or pri-miR-144.In both cases, bands of the appropriate size for the pre-miRNA wereobserved (FIG. 5B). In the case of pri-miR-451 processing the 5′ flankof the transcript folds into an additional hairpin, which may bereleased by Drosha to give additional fragments. As a result, only oneflank was observed. The identities of pre-miRNA bands were confirmed byNorthern blotting with oligonucleotide probes corresponding to thepredicted species (FIG. 5C). Considered together, these experimentsprovide both genetic and biochemical support for Drosha catalyzing theexcision of pre-miR-451 from its primary transcript.

Pre-miR-451 has an unusually short, 17 nt stem region. Previous studiesindicate that this is too short to be efficiently recognized andprocessed by Dicer (Siolas et al., 2004). We therefore examined the roleof Dicer in miR-451 maturation. We introduced the pre-miR-451 expressionvector into ES cells that are homozygous for Dicer conditional allelesand express Cre-ER. While acute Dicer loss caused a roughly 80-foldreduction in a control ES cell microRNA (miR-294), miR-451 levels didnot change (FIG. 5D). A pure population of continuous Dicer-null EScells showed more than a 500-fold reduction in conventional microRNA,whereas levels of miR-451 were unaffected (FIG. 2B). We also confirmedthis results using northern blot analysis of dicer nulls ES cellstransiently expressing the miR-451 precursor (FIG. 1A-B). Finally, weincubated synthetic miR-451 pre-miRNA with recombinant Dicer andobserved no mature cleavage products, though pre-let-7 was efficientlyprocessed. Thus, conversion of pre-miR-451 into a mature miRNA proceedsindependently of Dicer. We therefore strove to identify an alternativematuration pathway.

5.1.4 Ago2 Catalysis is Required for miR-451 Biogenesis

A By Northern blotting, we examined miR-451 species in wild-type andAgo2ADH mutant livers. This confirmed loss of the mature miRNA in themutant animals. However, we noted the appearance of an ˜40 nt band thatco-migrated with a synthetic pre-miR-451 and hybridized to probes to its5′ and 3′ arms (FIG. 6A). This indicated accumulation of the Droshacleavage product in mutant animals. Notably, the same bands seen intotal RNA were also detected in Ago2 immunoprecipitates (FIG. 6A). Thisdemonstrated the direct loading of the pre-miRNA into Ago2 and raisedthe possibility that the Ago2 catalytic center might help to catalyzethe maturation of this microRNA.

The well-established biochemical properties of Ago2 predict that itwould cleave a loaded pre-miR-451 after its 30th base. We searched forevidence of such an intermediate in fetal liver small RNA librariesencompassing an expanded size range. Plotting a size distribution ofreads corresponding to a conventional miRNA, miR-144, gave the expectedpattern, a sharp peak at ˜20 nt. In contrast, miR-451 showed aheterogeneous size distribution, exclusively because of variation at its3′ end. One abundant species corresponded precisely to the predicted Agocleavage product (FIG. 6B-C).

We confirmed the capacity of Ago2 to load and cleave pre-miR-451 usingin vitro assays (FIG. 5D). Wild-type or catalytically inactive Ago2complexes, or Ago1 complexes (FIG. 2B, 6D) were affinity purified from293T cells and mixed with 5′-end labeled pre-miR-451. Only wild-typeAgo2 produced the expected product, and this depended upon the presenceof Mg2+. No product was produced if we provided a mutant version of theprecursor in which a single point mutation disrupted pairing at thecleavage site. Beta elimination and ligation reactions confirmed thatcleavage left a free 3′ OH terminus as expected of Argonaute proteins.These data strongly support a role for the Ago2 catalytic center inmiR-451 maturation. This is perhaps akin to the proposed role ofpassenger strand cleavage in the maturation of siRISC.

Considered together, our results suggest a model in which miR-451 entersRISC through an alternative biogenesis pathway. Though Drosha cleavageproceeds normally, the Dicer step is skipped and the pre-miRNA is loadeddirectly into Argonaute. This is surprising, considering prior studiesindicating a coupling of Dicer cleavage and RISC loading Chendrimada, T.P. et al. Nature 436, 740-4 (2005), Wang, H. W. et al. Nat Struct MotBiol 16, 1148-53 (2009). Such a complex would also lack interactionsbetween the PAZ domain and the 3′ end of a conventional Dicer product.Song, J. J. et al. Nat Struct Biol 10, 1026-32 (2003), Wang, Y. et al.,Nature 456, 209-13 (2008). A prior report indicated the ability of RISCto accommodate such species and posited a potential for Ago cleavage inthe maturation of canonical microRNAs. Diederichs, S. & Haber, D. A.Cell 131, 1097-108 (2007). However, no physiological role for such anactivity was demonstrated, and we detect no measurable defects in theprocessing of canonical miRNAs in Ago2ADH mutants. MiR-451 maturationproceeds with Ago-mediated cleavage producing an intermediate that isfurther trimmed. While this could occur via either endo- orexo-nucleolytic digestion, the observed distribution of 3′ ends, manybearing single non-templated U residues, seems more consistent with thelatter model. Though the precise enzymology of this step remainsobscure, preliminary studies fail to support roles for Eri-1 or theexosome complex.

5.1.5 Methods Mouse Strains

Ago2 insertional mutant mouse strains, generated previously were usedfor mutant analysis, ES cell derivation and reporter analysis. Liu, J.et al. Science 305, 1437-41 (2004). Ago1 gene trap strain was generatedthrough germline transmission of Ago1 gene trap ES cells from BayGenomics (RRR031). Ago2 catalytically inactive mutant knock-in mice weregenerated through germline transmission of positive ES cell clonestargeted with Bacterial artificial chromosome (RP23-56M12) that has beenmodified with a point mutation D598A in the PIWI domain of Ago2.

Beta-Galactosidase Staining

For whole mount staining, embryos from different stages were dissectedtogether with their extra-embryonic compartments in PBS. Betagalactosidase staining was performed using millipore's stainingreagents. X-gal staining was performed overnight at room temperature.For placental sections, whole placentas were first stained for B-gal,sectioned and counterstained with Haematoxylin and Eosin.

Ago2 Mutant Crosses and Embryonic Stem (ES) Cell Derivation

Ago2 mutant phenotype was re-examined combining two insertional allelesfor ease of genotyping the homozygous progeny and to take advantage ofthe Ago2 beta gal reporter allele. Ago2 null ES cells were derived aspreviously described Nagy, A. et al., Manipulating the Mouse Embryo: ALaboratory Manual (CSHL press, 2003). Null cells were genotyped usingprimers specific to both insertional alleles. Null cells were genotypedusing primers specific to both insertional alleles. Ago2mc allele:forward (GACGGTGAAGAAGCACAGGAA) (SEQ ID NO: 22), reverse(GGTCCGATGGGAAAGTGTAGC) (SEQ ID NO: 23). Ago2gt allele: forward(ATGGGATCGGCCATTGAA) (SEQ ID NO: 24), reverse (GAACTCGTCAAGAAGGCG) (SEQID NO: 25).

RT-PCR, Western Blot and Immunoprecipitation

Ago2 RT-PCR primers were designed downstream of both insertionalalleles: Ago2F: TGTTCCAGCAACCTGTCATC (SEQ ID NO: 26),Ago2R:GATGATCTCCTGTCGGTGCT (SEQ ID NO: 27) Actin primers were used as anormalization control. ActinF: ATGCTCCCCGGGCTGTAT (SEQ ID NO: 28),ActinR: CATAGGAGTCCTTCTGACCCATTC (SEQ ID NO: 29). QRT-PCR was performedusing invitrogen superscript III and Applied biosystem cyber green PCRreagent. miRNA levels were measured using Applied Biosystems pri ormature miRNA assays. Ago2 western blot and immunoprecipitation analysiswere performed using abnova eif2c2 antibody (M01). P53 western wasperformed using santa cruz mouse monoclonal antibody (Pab240).

ES-Tetraploid Aggregation

Ago2 null ES cells were injected into tetraploid blastocyst aspreviously described Nagy, A. & Rossant, J. in Gene Targeting APractical Approach (ed. Joiner, A. L.) 189-192 (Oxford University Press,2000). Embryos were transferred to foster mothers and dissected atE12.5. Beta gal stained was performed as described above.

Peripheral Blood Collection and FACS Analysis

Blood was collected from decapitated fetuses (pre-mortem) usingheparanized microcapillaries and the CBC count was performed using thehemavet. For FACS analysis, single cells were isolated from neonatalliver, spleen and bone marrow and co-stained with Ter119 and CD71antibodies (BD) and analyzed on LSRII flow cytometer (BD) as previouslydescribed Socolovsky, M. et al. Blood 98, 3261-73 (2001). The Samenumber of events of each sample were collected according to doubletdescrimination gating and analyzed as follows: the ProE cell populationwas defined by CD71high/ter119 medium positive events. The ter119 highpopulation was further subdivided into basophilic, latebasophilic/polychromatic and orthochromatic/reticulocyte cellpopulations according to CD71 and FSC parameters to define thesubsequent differentiating erythroblasts Liu, Y. et al. Blood 108,123-33 (2006).

Small RNA Cloning and Bioinformatics Annotation

Total RNA was extracted from E18.5 livers using trizol. Two Small RNAlibraries with a size range of 19-30 nt and 30-40 nt were generatedusing a modified small RNA cloning strategy Aravin, A. & Tuschl, T. FEBSLett 579, 5830-40 (2005), Pfeffer, S. et al. Nat Methods 2, 269-76(2005). Briefly, the small RNA fraction was ligated sequentially at the3′OH and 5′phosphates with synthetic linkers, reverse transcribed andamplified using solexa sequencing primers. Around 7 million reads weregenerated for each small RNA library. Sequences were then trimmed fromthe 3′ linker, collapsed and mapped to the mouse genome with nomismatches using multiple annotation tracks, namely: UCSC genes, miRNAsand repeats. For this study we used the mirbase database to annotate thecloned miRNAs.

Cell Culture, Plasmids, Transfections and Sensor Assays

Mir-144-451 expression vector was constructed by cloning the genomiccluster into pMSCV retroviral vector. Cre-ER MEFs and ES cells werecultured as previously described48. Excision of dicer and drosha allelewas mediated through tamoxifen treatment (100 nM) for 5 days followed bytransient transfection of miR-451 expressing plasmid using lipofectamine(Invitrogen). For in vitro procesessing assays and northern blots 293Tcells were cultured in DMEM+10% FBS and cotransfected using LT-1 Minisreagent with flag tagged drosha and DGCR8 constructs, myc tagged Ago2 orAgo1 with MSCV-miR144-451 expression vector or myc tagged Ago2 alone.Dual luciferase assays were performed as previously described. Forvalidation of the Ago2 null ES cells, a luciferase plasmid with noartificial site was cotransfected with a perfectly matched siRNA duplex(dharmacon).

Drosha In-Vitro Processing Assays

PCR fragment mapping to miR-451 and mir-144 were amplified out of thehuman genome with T7 promoter sequence. Pri-451 and Pri-144 RNAtranscripts were generated using the genomic PCR product and Ambion's T7in-vitro transcription kit. Transcripts were gel purified and used in adrosha in-vitro processing assay as previously described Lee, Y. et al.Nature 425, 415-9 (2003), Denli, A. M., et al. Nature 432, 231-5 (2004).

RNA Northern Blot Analysis

RNA was extracted from liver homogenates and Ago2 immunoprecipitates(IPs) using trizol reagent. 10-15 ug of total RNA and ½ of the IPed RNAwas run a 20% acrylamide gel and transferred onto a positively chargednylon membrane (hybond). Membranes were crosslinked, prehybridized inultra-hyb solution (ambion) and hybridized with P32 labeled DNA probescomplementary to miR-451 and let-7c. Membranes were washed with 2×SSC,0.1% SDS and 1×SSC, 0.1% SDS and exposed on a phosphoimager screenovernight.

Ago2 Cleavage Assays and Beta Elimination

Ago2 myc tagged constructs (wt and D797A) were transfected in 293Tcells. Lysates were collected after 48 hours, immunoprecipitated usingmyc agarose beads. The catalysis reaction was carried out on beads using5′ P32 end labeled synthetic pre-miR-451 (dharmacon) as previouslydescribed (Liu, J. et al. Science 305, 1437-1441(2004)). Betaelimination was performed through treating the purified RNA from theAgo2 beads with Sodium periodate for 30 min at room temperature followedby ethanol precipitation. The RNA was resuspended in loading buffercontaining TBE and run on a 20% acrylamide gel where the betaelimination reaction occurs.

5.2 Example 2: Design of miR-451 shRNA Structural Mimics

To investigate sequence versus structural requirements for entry intothe alternative miRNA biogenesis pathway, we engineered shRNAs asstructural mimics of the miR-451 precursor to produce let-7c. Thisstructurally designed shRNA was at least as efficient as the nativepre-let-7c in suppressing a GFP or luciferase reporter containingperfect let-7 complementary sites (FIG. 1A-C) We also designed a miR-451shRNA mimic targeting p53, here targeting the following site in the p53mRNA: UCCACUACAAGUACAUGUGUAA (SEQ ID NO: 6). (FIG. 1D, FIG. 3A-FIG. 3B).Accordingly, an expression construct can therefore be used toefficiently repress p53 in cells by expressing the mir-451 shRNA mimic.

5.2.1 Methods

Testing the functionality of miR-451 mimics was performed using threestrategies: (1) cotransfection of let-7-miR-451 mimics, pre-let-7 orlet-7 duplex or CTRL RNAs (dharmacon) at a 100 nM concentration withlet-7c luciferase reporter construct containing two perfect matchingsites in the 3′UTR in HEK293 cells, (2) Similarly, tetracyclineinducible Let-7 GFP sensor ES cells containing two perfectly matchedsites cotransfected with PE-labeled siRNA and let-7-miR-451 mimics (50nM) followed by GFP analysis of PE positive cell population using LSRIIflow cytometer (BD). GFP sensor was induced using dox (1 ug/ml), (3) Forp53 knockdowns, ES cells were transfected with p53 shRNA and p53-miR-451mimics followed by p53 induction using adriamycin (0.5 ug/ml) within thelast 8 hours before harvest. All cells were harvested 48 hourspost-transfection.

5.3 Example 3: Expression of miR-451 shRNA Structural Mimics

The miR-451 shRNA mimics described in the present application provide atool with broad applicability for use in RNAi based applications, bothfor research and in medical applications. As a non-limiting example,miR-451 shRNA mimics designed through the methods of the presentapplication and targeted against particular genes can be used toefficiently repress expression of these genes in mammalian cells, bothin culture and in whole animals, including transgenic animals, byexpression of the miR-451 shRNA mimic or a precursor molecule for suchshRNA. As a further non-limiting example, an MSCV expression plasmid(see for example, FIG. 2A-FIG. 2B, Ex. 1), is used to illustrate how tomake expression constructs encoding miR-451 shRNA mimics targetedagainst p53, based on a miR-451 backbone. The approach is outlined inFIG. 7.

A mir-144-451 fragment cloned in the MluI/BglII site of the Mir-144-451expression vector (FIGS. 2A-2B, 7, Ex. 1) and encompassing themir-144-455 cluster sequence is amplified out of the human genome. Fromthe amplified fragment, a miR-451 cassette is generated by subcloning afragment of the mir-144-451 cluster sequence comprising 5′ and 3′miR-451 flanking sequences, engineering restriction sites on each of the5′ and 3′ ends of that fragment, and subcloning the resulting cassetteinto an MSCV expression plasmid backbone. An MSCV expression constructencoding a miR-451 shRNA mimic targeted against p53 mRNA is generated byreplacing the native miR-451 precursor sequence (FIG. 7, shaded portion;AAACCGTTACCATTACTGAGTTTAGTAATGGTAATGGTTCT) (SEQ ID NO: 11) with asequence encoding the mir-451 shRNA mimic. For convenience, it may bedesirable to engineer restriction sites into such construct at the 5′and 3′ ends of the sequence encoding the miR-451 shRNA mimic, such thatan alternative miR-451 shRNA mimic may be easily integrated into theconstruct by removing the p53 targeted mir-451 shRNA mimic sequence andreplacing that sequence with that of the desired targeted miR-451 shRNAmimic.

Generally, so that sufficient cis-acting sequences (and structuraldeterminants) are retained in the expressed miR-451 shRNA mimic to allowfor efficient Drosha processing, it is appropriate to include 20 or morebp of miR-451 flanking sequence. In some instances it may be desirableto alter the length of the flanking sequence to optimize expression ofthe mature miR-451 mimic. Any one of various lengths of either or both5′ and 3′ flanking sequence from 5 to 60 bp may be selected and theconstruct engineered so as to integrate the desired length of flankingsequence into the expression construct cassette. One of skill in the artwill appreciate that such lengths of flanking sequence include 5 bp and60 bp and each intervening integer value between 5 and 60.

The examples are provided to illustrate the general utility of theinvention and are not meant to limit the implementation of thisapproach. The approach illustrated here offers considerable flexibilityin use of various expression constructs, alternative vectors anddelivery methods, all of which may be routinely optimized for use inparticular cells, tissues, organs or animals. For example, expressionconstructs employing a miR-451 backbone for expression of the mniR-451shRNA mimics of the invention can be based on any analogous RNA polII-based expression constructs used for expression of conventionalshRNAs, including constructs incorporating inducible/repressible,tissue-specific or developmentally regulated promoters, IRES sites forbicistronic expression, selectable markers, fluorescent markers and RNAisensors.

5.4 Example 4: MicroRNA-451 Based shRNA Precursors (Drosha Products) areFunctional

MicroRNA-451 based shRNA precursors (drosha products) are functional inmouse ES cells and manifest a different dose response compared to miR-30based shRNAs precursor mimics. Without wishing to be bound to, orlimited by, any scientific theory, this may suggest different rules intarget recognition between the two pathways. Titration curves showingthe efficiency of p53 specific shRNAs in miR-451 and miR-30 based mimicswere generated (FIG. 8). Three mouse p53 shRNA synthetic RNAs(shp53.1224, shp53.279 and shp53.1404) in miR-451 (40 nt long) andmiR-30 (61 nt long) precursor structures were transfected in mouse EScells. p53 hairpin potency is primarily ranked as best, intermediate andweak according to sensor data described on miR-30 based shRNAs inprimary vectors expression system (Fellmann, C. et al. Mol Cell 41,733-746 (2011)). Concentrations of the p53 shRNAs were titrated using asimilar length control shRNA as a control to insure equal amount of thetransfected RNA at each concentration of the targeting shRNA. Cells weretreated with doxorubicin at final concentration of 500 ng/ml for 8 hrsbefore harvest. p53 expression level was detected by western blot andquantified based on negative control.

5.5 Example 5: Primary MicroRNA-451 Based shRNA is Functional

Stable expression of the miR-451 mimics using a miR-451 backbone wasaccomplished as has been described for miR-30. The miR-451 pathwaydepends on Drosha and Ago2 processing only independently of Dicer.Measurement of knockdown efficiency of miR-451 and miR-30 based primarymimics was performed. (FIGS. 9A and B) p53 Western blots (left panel)followed by quantification (right panel) on primary MEFs infected at lowMultiplicity of Infection (MOI) “single copy” and NIH3T3 cells infectedat low or high MOI with mouse p53 shRNAs in miR-451 or miR-30 retroviralbackbones (MSCV), respectively. Cells were treated with doxorubicin atfinal concentration of 500 ng/ml for 8 hrs. (FIG. 9C) Renilla luciferaseknockdown (left panel) using Four Renilla luciferase shRNAs in miR-451or miR-30 retroviral backbones (MSCV) were infected in NIH3T3-renillareporter cells. Renilla luciferase luminescence was normalized to totalprotein absorbance using BCA measuring assay. GFP expression level(infection efficiency) was shown in the lower graph.

5.6 Example 6: Primary Micro-451 Based shRNAs are Processed Through themiR-451 Pathway

Northern blot analysis from matching RNA samples of the p53 experimentsin Example 5 (FIG. 9A-FIG. 9C) was performed using radio labeled probescomplementary the mature 22 nt sequences of processed shRNAs. Mature p53shRNAs were detected in NIH3T3 cells infected with pri-shp53-miR-451mimics and pri-shp53-miR-30 mimics at low (FIG. 10A) or high (FIG. 10B)MOI. Densitometry quantifications are shown in right panels. (FIGS. 10Cand D) Ago2 immunoprecipitation-northern analysis using probes specificto the mature 22 nt sequences of processed primary shRNAs.Pri-shp53-miR-451 and pri-shp53-miR-30 mimics were transfected alone(FIG. 10C) or co-transfected with wild type Ago2 or Ago2 catalytic deadconstructs (FIG. 10D) into HEK293T cells. miR-451 mimics weresuccessfully loaded into Ago2 complexes but only processed to theirmature form in the wild type protein. Precursor 40mer mimic accumulatedin the catalytically inactive Ago2 (FIG. 10).

5.7 Example 7: miR-451 shRNA Structural Mimic Design Steps

1. choose 22mer target sequence for example Renilla-shRNA-1 (the firstone we tested)

TAGGAATTATAATGCTTATCTA (SEQ ID NO: 30)

2. Reverse complement Renilla 22mer target sequence

TAGATAAGCATTATAATTCCTA (SEQ ID NO: 31)

3. trim 1 through 18 nt in the reverse complement

TAGATAAGCATTATAATT (SEQ ID NO: 32)

4. reverse complement the trimmed 1-18 to make the stem

AATTATAATGCTTATCTA (SEQ ID NO: 33)

5. join sequences from step 2 and step 4 (the stem) in this order togenerate the 40mer shRNA

(SEQ ID NO: 34) TAGATAAGCATTATAATTCCTA AATTATAATGCTTATCTA

6. To make sure there is a bulge at the first position: If the firstnucleotide of shRNA is A or T make sure that the 40^(th) position is C(like endogenous miR-451), if the first position is a C or G, the40^(th) position should be changed to an A. In this case substitute A atposition 40 with C:

(SEQ ID NO: 35) TAGATAAGCATTATAATTCCTA AATTATAATGCTTATCT C

7. Add flanking regions of endogenous miR-451 and restriction sites forcloning into the destination vector (the minimal backbone depicted herein lowercase letters is about 61 nt and 63 nt long for the 5′ and 3′flanks respectively)

(SEQ ID NO: 36) gaagctctctgctcagcctgtcacaacctactgactgccagggcacttgggaatggcaaggTAGATAAGCATTATAATTCCTAAATTATAATGCTTATCTCtcttgctatacccagaaaacgtgccaggaagagaactcaggaccctgaa gcagactactggaa

Restriction sites are either introduced with PCR primers to generate theDNA duplex for cloning or two complementary oligos are ordered andannealed to make the duplex (FIGS. 11A-11C and 12).

shRNA targeted to other genes can be made in an analogous manner, usinga difference sequence of nucleotides to result in the shRNA as describedin the various embodiments herein.

5.8 Example 8: Knockdown of Long Non-Coding RNA

An example of using HOTAIR shRNA miR-451 and miR-30 mimic design in MSCVexpression vector. Target sequences were chosen from siRNAs reported inthe literature to target HOTAIR efficiently (Wan, Y. and Chang, H. Y.Cell Cycle 9, 3391-3392 (2010)). When tested in culture, shRNA andsiRNAs behave differently. miR-451 based mimics successfully knockdownlevels of HOTAIR in one case more efficiently that miR-30 based mimics.

5.9 Example 9: miR-451 Tiling Chip to Generate a miR-451 Based shRNALibrary

In order to understand the underlying rules of processing and targetrecognition of miR-451 mimics, we generated an shRNA tiling chip at onenucleotide step for 10 different genes (p53, bcl2, mcl1, myc, rpa3,kras, PCNA, GFP, mkate2 and mcherry). 164 mer long synthetic oligolibrary was generated using Agilent's (Santa Clara, Calif., USA) oligosynthesis platform. The library was then amplified using the constantflanks and cloned in an MSCV destination vector. The library was thentransfected in 293T cells and the processing of the shRNAs was analyzedthrough the generation of small RNA libraries followed by solexasequencing. The quality of these libraries and their processingefficiency are analysed. Table 1. depicts the efficiency of thesequences recovered in the small RNA library according to input.

TABLE 1 Efficiency of Sequences recovered in the small RNA Library shRNAtotal genome library number mapping mapping of reads reads reads miRNAmiR-451 mature 11,761,255 67.59% 1.16% 55.12% alignment total RNA to19-33nt predicted Ago2 IP 15,293,024 79.35% 1.67% 77.62% CHIP- matureshRNAs 19-33nt pre-fraction 2,285,916 19.64% 0.66% 0.08% total RNA 40 ntmiR-30 mature 13,824,773 62.81% 7.42% 53.08% alignment total RNA onAgo2IP 15,640,560 64.02% 9.49% 61.74% predicted mature 22mer guidestrands

1-27. (canceled)
 28. The shRNA of claim 28 having the structure

wherein X₂ to X₂₂ are nucleotides complementary to a sequence in atarget gene, and are in a sequence other than the mature sequence ofmiR-451; Y₄ to Y₂₀ are nucleotides complementary to X₂ to X₁₈; andwherein, X₁ and Y₃ are not complementary.
 29. An expression vectorcomprising a sequence encoding the shRNA according to claim 28 operablylinked to an RNA polymerase promoter.
 30. A library of expressionvectors, each expression vector encoding the shRNA according to claim 28operably linked to an RNA polymerase promoter.
 31. An isolated mammaliancell comprising the shRNA according to claim
 28. 32. A transgenic mammalwhose genome comprises a sequence encoding the shRNA according to claim28.
 33. A method of attenuating expression of a target gene in amammalian cell, the method comprising introducing into the mammaliancell an expression vector comprising a sequence encoding the shorthairpin RNA molecule (shRNA) of claim 28, wherein the shRNA molecule isexpressed in the mammalian cell in an amount sufficient to attenuateexpression of the target gene in a sequence specific manner, wherebyexpression of the target gene is inhibited.